Astaxanthin synthase

ABSTRACT

The present invention is directed to genetic materials useful for the preparation of astaxanthin from beta-carotene, such as polypeptides having astaxanthin synthase activity, DNA fragments coding for astaxanthin synthase, recombinant organisms and the like. Those novel genetic materials may be derived from  Phaffia rhodozyma . The present invention also provides a process for the production of astaxanthin.

The present invention relates to recombinant production of carotenoidsand biological materials useful therefor.

Phaffia rhodozyma (P. rhodozyma) is a carotenogenic yeast strain whichproduces astaxanthin. Astaxanthin is distributed in a wide variety oforganisms such as animals (birds such as flamingo and scarlet ibis, andfish such as rainbow trout and salmon), algae and microorganisms. It isalso recognized that astaxanthin has a strong antioxidation propertyagainst oxygen radicals, and is expected to be useful pharmaceuticallyfor protecting living cells against certain diseases, such as a cancer.Moreover, industrial need for astaxanthin as a coloring reagent isincreasing, especially in the industry of farmed fish like salmon,because astaxanthin imparts a distinctive orange-red coloration to theanimals and contributes to consumer appeal in the marketplace.

P. rhodozyma is known as a carotenogenic yeast strain which producesastaxanthin. Different from the other carotenogenic yeast, Rhodotorulaspecies, P. rhodozyma can ferment some sugars such as D-glucose. This isan important feature from a viewpoint of industrial application. In arecent taxonomic study, a sexual cycle of P. rhodozyma was revealed andits telemorphic state was designated under the name of Xanthophyllomycesdendrorhous (W. I. Golubev; Yeast 11, 101-110, 1995). Some strainimprovement studies to obtain hyper producers of astaxanthin from P.rhodozyma have been conducted, but such efforts have been restricted toemploy the method of conventional mutagenesis and protoplast fusion inthis decade. Recently, Wery et al. developed a host vector system usingP. rhodozyma in which a non-replicable plasmid was integrated inmultiple copies into the genome of the ribosomal DNA of P. rhodozyma(Wery et al., Gene, 184, 89-97, 1997). Verdoes et al. reported moreimproved vectors to obtain a transformant of P. rhodozyma as well as itsthree carotenogenic genes which code for the enzymes that catalyze thereactions from geranylgeranyl pyrophosphate to beta-carotene (WO97/23633).

A specific biosynthetic pathway for carotenogenesis branches from thegeneral isoprenoid pathway at the point of an important intermediate,farnesyl pyrophosphate (FPP) (FIG. 1). FPP and IPP are condensed bygeranylgeranyl pyrophosphate (GGPP) synthase which is encoded by crtE inP. rhodozyma to produce GGPP. GGPP is then converted to beta-carotene bythe sequential reaction of an enzyme functioning doubly as phytoenesynthase and lycopene cyclase which is encoded by crtBY and phytoenedesaturase encoded by crtI.

In bacteria, enzymes and genes which are involved in xanthophyllformation have been isolated and characterized in detail. Beta-carotenehydroxylase which is coded by crtZ is involved in the two steps ofhydroxylation for the beta-ionone-ring of beta-carotene at both of theends. The crtZ gene has been cloned from a wide variety of organismssuch as Erwinia uredovora (Misawa et al., J. Bacteriol., 172, 6704-6712,1990), Flavobactor species (L. Pasamontes et al., 185 (1), 35-41, 1997)and Agrobacterium aurantiacum (Misawa et al., J. Bacteriol., 177 (22),6575-6584, 1995). Beta-carotene ketolase which is encoded by crtWcatalyzes the two steps of introduction of an oxo-group into thebeta-ionone -ring of beta-carotene at both of the ends. Kajiwara et al.cloned and sequenced the bkt gene corresponding to crtW in eubacteriafrom Haematococcus bluvialis (Kajiwara et al., P. Mol. Biol., 29,343-352, 1995). Harker et al. also cloned and sequenced the crtO genecorresponding to crtW in eubacteria from Synechococcus PCC7942 (Harkeret al., FEBS Letters, 404, 129-134, 1997). Both enzymes, i.e., thehydroxylase and the ketolase, have wide substrate specificity and thisensures the formation of a wide variety of xanthophylls in case both ofthe enzymes react at the same time, depending on the reaction condition.(FIG. 1)

As described above, all the genes which were involved in the formationof beta-carotene from FPP have been isolated but the enzymes and geneswhich would be involved in the last step of xanthophyll formation frombeta-carotene have not been identified on the protein and DNA level inP.rhodozyma. Although Johnson et al. (Crit. Rev. Biotechnol, 11 (4),297-326, 191) proposed the existence of two independent pathways forastaxanthin formation by assuming that some of the xanthophyll compoundsisolated by them would be intermediates of astaxanthin biosynthesis,these two independent pathways could not be proven because enzymes andgenes which are involved in such pathways could not be isolated.Furthermore, it can not be excluded that these xanthophyll compoundscould have resulted from an experimental artifact in the isolation stepof these compounds. Failure to isolate a mutant from P. rhodozyma whichaccumulates intermediates in the biosynthetic pathway from beta-caroteneto astaxanthin made it difficult to clarify the biosynthetic pathwayfrom beta-carotene to astaxanthin.

SUMMARY OF THE INVENTION

This invention relates to a gene and an enzyme which is involved in thelast step of astaxanthin biosynthesis (i.e., from beta-carotene toastaxanthin).

The present invention provides an isolated DNA, for example, a cDNAincluding a nucleotide sequence coding for astaxanthin synthase which isinvolved in the reaction from beta-carotene to astaxanthin in P.rhodozyma, like the AST gene.

In a preferred embodiment, the cloned DNA fragment can be characterizedin that

(a) the nucleotide sequence encodes an enzyme having the amino acidsequence described in SEQ ID NO: 1, or

(b) the nucleotide sequence encodes a variant of the enzyme selected in(a), which nucleotide sequence is either (i) an allelic variant or (ii)an enzyme having one or more amino acid insertions, deletions, and/orsubstitutions and having the stated enzyme activity.

In another preferred embodiment, the isolated cDNA fragment can bederived from a gene of Phaffia rhodozyma and is selected from:

(i) a cDNA sequence represented by SEQ ID NO: 2;

(ii) an isocoding or an allelic variant for the cDNA sequencerepresented by SEQ ID NO: 2; and

(iii) a derivative of a cDNA sequence represented by SEQ ID NO: 2 withinsertions, deletions, and/or substitutions of one or morenucleotide(s), and encoding a polypeptide having the enzyme activity.

In another preferred embodiment, the present invention includes theisolated cDNA as described above, which is characterized in that thenucleotide sequence is:

(i) a nucleotide sequence represented in SEQ ID NO: 2;

(ii) a nucleotide sequence which, because of the degeneracy of thegenetic code, encodes an astaxanthin synthase having the same amino acidsequence as that encoded by the nucleotide sequence in (i); and

(iii) a nucleotide sequence which hybridizes to the complement of thenucleotide sequence from i) or ii) under standard hybridizingconditions.

In still another preferred embodiment, an isolated genomic DNA fragmentcan be derived from a gene of Phaffia rhodozyma and is selected from:

(i) a genomic DNA sequence represented by SEQ ID NO: 3;

(ii) an isocoding or an allelic variant for the genomic DNA sequencerepresented by SEQ ID NO: 3; and

(iii) a derivative of a genomic DNA sequence represented by SEQ ID NO: 3with insertions, deletions, and/or substitutions of one or morenucleotide(s), and coding for a polypeptide having the enzyme activity.

In another preferred embodiment the present invention includes theisolated genomic DNA as described above, which is characterized in thatthe nucleotide sequence is:

(i) a nucleotide sequence represented in SEQ ID NO: 3;

(ii) a nucleotide sequence which, because of the degeneracy of thegenetic code, encodes an astaxanthin synthase having the same amino acidsequence as that encoded by the nucleotide sequence in (i); and

(iii) a nucleotide sequence which hybridizes to the complement of thenucleotide sequence from i) or ii) under standard hybridizingconditions.

Another aspect of the present invention is a recombinant polypeptidehaving astaxanthin synthase activity and which is involved in thereaction from beta-carotene to astaxanthin in P. rhodozyma which isobtainable by the expression of the cloned DNA fragment as set forthabove.

A preferred embodiment of the recombinant polypeptide of the presentinvention is characterized in that

(a) the polypeptide has an amino acid sequence as described in SEQ IDNO: 1, or

(b) the polypeptide is a variant of the peptide defined in (a) which isselected from (i) an allelic variant or (ii) an enzyme having one ormore amino acid insertions, deletions and/or substitutions and havingthe stated enzyme activity.

The present invention also includes variants of the polypeptides setforth above. Such variants are defined on the basis of the amino acidsequence of the present invention by insertions, deletions, and/orsubstitutions of one or more amino acid residues of such sequenceswherein such variants still have the same type of enzymatic activity asthe corresponding polypeptides of the present invention or they are theresult of the well known phenomenon of allelic variation. Suchactivities can be measured by any assays known in the art orspecifically described herein. Such variants can be made either bychemical peptide synthesis known in the art or by recombinant means onthe basis of the DNA sequences as disclosed herein by methods known inthe state of the art, such as, e.g. that disclosed by Sambrook et al.(Molecular Cloning, Cold Spring Harbour Laboratory Press, New York, USA,second edition 1989).

Amino acid exchanges in proteins and peptides which do not generallyalter the activity of such molecules are known in the state of the artand are described, for example, by H. Neurath and R. L . Hill in “TheProteins” (Academic Press, New York, 1979, see especially FIG. 6, page14). The most commonly occurring exchanges are: Ala/Ser, Val/Ile,Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe,Ala/Pro, Lys/Art, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly as well asthe reverse. It is also possible to add or delete one or several aminoacid residues(s) at N- and/or C-terminal of the enzyme without anycritical effect on the activity of the present synthase.

Furthermore, the present invention is not only directed to the DNAsequences as disclosed e.g., in the sequence listing as well as theircomplementary strands, but also to those which include these sequences,DNA sequences which hybridize under standard conditions with suchsequences or fragments thereof and DNA sequences, which because of thedegeneration of the genetic code, do not hybridize under standardconditions with such sequences but which code for polypeptides havingexactly the same amino acid sequence.

The said enzyme activity is expressed as the enzyme activity whichrenders astaxanthin production to beta-carotene producing microorganismby means of the transformation to express its corresponding gene in thesaid beta-carotene producing host organisms.

The said enzyme activity is also expressed as the enzyme activity whichrenders astaxanthin production to microorganism which accumulatesintermediate xanthophyll from beta-carotene to astaxanthin described inFIG. 1; e.g. echinenone, beta-cryptoxanthin, canthaxanthin,3′-hydroxyechinenone, 3-hydroxyechinenone, zeaxanthin, phoenicoxanthinand adonixanthin.

The said enzyme activity can also be expressed as the enzyme activitywhich catalyzes the astaxanthin formation from various substrates suchas beta-carotene, echinenone, beta-cryptoxanthin, canthaxanthin,3′-hydroxyechinenone, 3-hydroxyechinenone, zeaxanthin, phoenicoxanthinand adonixanthin under appropriate in vitro condition which constitutesof membrane fraction such as natural membrane like microsome andartificial membrane like liposome in company with appropriate electrondonor like NADPH.”

In the present invention, unless otherwise indicated, the hybridizationreactions are generally carried out at 42° C., which is 15 to 35° C.below the T_(m) of most DNA probes, thus ensuring a maximum rate ofhybridization. The desired stringency of hybridization is achieved bywashing, e.g., the filter at a salt concentration and temperature thatis approximately 5 to 15° C. below the T_(m) for a perfectly matchedhybrid. The salt concentration and temperature, however, may be adjustedto less stringent conditions if significant mismatching of sequence isexpected (e.g., when probing for the same gene in a different species orfor a different but related sequence).

As used herein, the phrase “standard conditions” for hybridization meansthe conditions which are generally used by a person skilled in the artto detect specific hybridization signals and which are described, e.g.by Sambrook et al., (s.a.) or preferably so-called stringenthybridization and non-stringent washing conditions, or more preferablyso-called stringent hybridization and stringent washing conditions aperson skilled in the art is familiar with and which are described,e.g., in Sambrook et al. (s.a) or more preferably so-called mediumstringent conditions, e.g. using the DIG (digoxigenin) labeling kit andluminescent detection kit of Boehringer Mannheim (Mannheim, Germany)following the protocol given by the manufacturer and using as thehybridization solution:

formamide (WAKO, Osaka, Japan) 50% (V/V)

5×SSC

blocking reagent (Boehringer) 2% (W/V)

N-lauroylsarcosine 0.1% (W/V)

SDS 0.3% (W/V)

at a temperature of 42° C. over night and subsequently washing anddetection as indicated by the manufacturer.

For example, a typical wash sequence includes washing the hybridizedblot first with a solution A containing 2×SSC/0.1% SDS in water at roomtemperature. Next, the blot is washed twice in solution B containing0.1×SSC/0.1% SDS in water at a temperature to be determined based on thedesired level of stringency. For example, a perfectly matched hybrid maybe washed at a temperature from about 55° to about 65° C.; for a probefrom a related gene or from a different species, the wash temperaturemay be, for example, from about 37° C. to about 52° C. Unless otherwiseindicated, this washing condition was used in the present invention.

DNA sequences which are derived from the DNA sequences of the presentinvention either because they hybridize with such DNA sequences (seeabove) or can be constructed by the polymerase chain reaction by usingprimers designed on the basis of such DNA sequences can be preparedeither as indicated, namely by the PCR reaction, or by site directedmutagenesis (see e.g., Smith, Ann. Rev. Genet. 19, 423 (1985)) orsynthetically as described, e.g., in EP 747 483 or by the usual methodsof Molecular Cloning as described, e.g., in Sambrook et al. (s.a.).

The present invention also includes a vector or plasmid that contains aDNA as described above and a host cell transformed or transfected by aDNA as described above or a vector or plasmid as indicated above.

The present invention also provides a recombinant organism which isobtainable by the transformation of a host using a recombinant DNAcarrying the DNA as mentioned above.

The present invention also includes a method for producing an enzymaticpolypeptide capable of catalyzing the reaction from beta-carotene toastaxanthin, which includes culturing a recombinant organism describedabove under conditions conductive for the production of the enzymaticpolypeptide.

In a further aspect, the present invention provides a method for theproduction of astaxanthin which includes introducing one or more of theDNAs described above into an appropriate host organism and cultivatingthis transformed organism under conditions conductive for the productionof astaxanthin.

The enzymatic polypeptide of the present invention is also useful in amethod for producing astaxanthin, which method includes contactingbeta-carotene with a recombinant polypeptide having an astaxanthinsynthase activity involved in the reaction from beta-carotene toastaxanthin as set forth above in the presence of an appropriateelectron donor in an appropriate reaction mixture containing anappropriate reconstituted membrane. In this method, the recombinantpolypeptide may be present in the form of a reconstituted membrane whichis prepared from biological membranes such as, for example, microsomesor mitochondrial membranes. The recombinant polypeptide may be alsopresent in the form of a reconstituted artificial membrane, such as forexample, a liposome. An electron donor, such as, cytochrome P450reductase is an example of an appropriate electron donor which canreduce a reaction center of the enzyme of the present invention.

Another embodiment of the invention is an isolated polynucleotideencoding a polypeptide which is SEQ ID NO:1, an isolated polynucleotidewhich is SEQ ID NO:2, or an isolated polynucleotide which is SEQ IDNO:3.

Another embodiment of the invention is a polypeptide having astaxanthinsynthase activity which is SEQ ID NO:1. The present invention alsoincludes a vector containing a polynucleotide which encodes SEQ ID NO:1,a polynucleotide which is SEQ ID NO:2, or a polynucleotide which is SEQID NO:3. A host cell is also provided which is transformed with thevector set forth above.

In another embodiment, the present invention provides a process forproducing astaxanthin which includes: (a) cultivating in a suitableculture medium a recombinantly produced host cell containing apolynucleotide which encodes a polypeptide having astaxanthin synthaseactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are included to further illustrate the presentinvention together with the detailed description given below.

FIG. 1 shows the biosynthetic pathway from acetyl-CoA to astaxanthin inP. rhodozyma.

FIG. 2 shows a restriction map of the plasmid pR6 which harbors apartial genomic AST gene.

FIG. 3 shows an expression study for the AST gene to which a 6×His tagwas added at its amino terminal end on removal of its transmembranedomain. The cells from 0.1 ml of broth were subjected to 10 % sodiumdodecyl sulfide—polyacrylamide gel electrophoresis (SDS-PAGE). Lane 1,Molecular weight marker (105 kDa, 82.0 kDa, 49.0 kDa and 33.3 kDa, up todown, Bio-RAD, Richmond, U.S.A.); Lane 2, E. coli (BL21 (DE3) (pLysS)(pAST315) without IPTG); Lane 3, E. coli (BL21 (DE3) (pLysS) (pAST315)with 1.5 mM IPTG); Lane 4, molecular weight marker).

DETAILED DESCRIPTION OF THE INVENTION

In general, there are a number of methods to clone a gene encodingbiosynthetic enzymes. For example, degenerate PCR can be used.Degenerate PCR is a method to clone a gene of interest which has highhomology of its encoded amino acid sequence to that of a known enzymefrom other species which have the same or similar function. A degenerateprimer, which is used as a set of primers in degenerate PCR, wasdesigned by a reverse translation of the amino acid sequence tocorresponding nucleotides (“degenerated”). In such a degenerate primer,a mixed primer which consists of any A, C, G or T, or a primercontaining inosine at an ambiguity code is generally used. After cloningof a partial fragment of the gene of interest the genetic fragmentcontaining the entire gene can be screened using the cloned and labeledpartial DNA fragment as a probe.

In the case of cloning a gene encoding an enzyme whose activity can bemeasured by an enzymatic assay, purification of such an enzyme bymonitoring enzyme activity and determination of its amino acid sequencefor the enzyme is a good method. An amino acid sequence thus obtained iseasily translated in reverse into the corresponding nucleotidesequence(s). A DNA fragment which has the corresponding nucleotidesequence can be synthesized iii vitro with a DNA synthesizer and labeledfor direct usage as a hybridization probe. An alternative way to obtaina hybridization probe is the degenerate PCR method using the amino acidsequence information.

To clone a gene whose function can not be characterized enzymatically, amethod called shot-gun screening has been employed as a conventionalcloning method. This method includes the isolation of a mutant strainwhich lacks the specific gene coding for any of the biosynthetic enzymesof interest, (and transformation of the mutant strain by the DNAprepared from the organism that has an intact gene which corresponds tothe gene as such mutated). For an isolation of such a mutant,conventional mutagenesis is often used. Confirmation of the acquiredphenotype which is the same as that of the parent strain can beperformed by examination of its auxotrophy and the like. In the casethat the donor DNA contains the gene which corresponds to the mutatedgene in the mutant strain, the transformant by such a gene acquired thesame phenotype as the parent strain as a result of geneticcomplementation.

As a vector, any form of vectors whether they can replicate or not inthe cloning host, can be used for shot gun cloning. For the usage of areplicative vector, the capability of complementation is a requisite andit is not necessary that such a vector contain a homologous sequence tothe genome of the recipient. In the case of using a non-replicativevector, it is necessary that such a vector contain a homologous sequenceto the genome of the host to make a recombination between donor andrecipient DNAs.

For cloning of the DNAs of the present invention the method called“color complementation” can be employed. Non carotenogenic organisms,such as Escherichia coli can acquire the carotenogenic ability as aresult of transformation by carotenogenic genes which could be clonedfrom carotenogenic organisms such as Erwinia uredovora, Erwiniaherbicola and the like. E. coli harboring crtE, crtB, crtI and crtY canproduce beta-carotene and color the cells yellow. Exploiting such acharacteristic, a number of members of the carotenogenic gene familyhave been cloned from various carotenogenic organisms such as bacteriaand plants. For example, to clone the crtY gene coding for lycopenecyclase, E. coli harboring crtE, crtB and crtI on a compatible vectoragainst pUC vector is prepared as a transformation host. Such a hostturns red which shows accumulation of lycopene. Next, a cDNA or genomiclibrary from carotenogenic organisms can be constructed using the pUCvector. If the gene corresponding to crtY in the donor carotenogenicorganism is present in the transformed plasmid, genetic complementationwould occur and the E. coli cells would turn yellow which would show theacquisition of the ability to produce beta-carotene. In fact, crtE,crtBY and crtI genes were cloned from P. rhodozyma by this method(Verdoes et al., WO 97/23633).

Regarding the cloning of the gene which is involved in the reaction frombeta-carotene to astaxanthin, Kajiwara et al. constructed a cDNAexpression library from P. rhodozyma in the host E. coli harboring crtE,crtB, crtI and crtY genes from E. uredovora (Kajiwara et al., WO96/28545, 1996). In such a cloning system, a gene which is involved inthe reaction from beta-carotene to astaxanthin could be theoreticallycloned from P. rhodozyma by judging red pigmentation which shows theaccumulation of canthaxanthin or astaxanthin. However, such a gene hasnot been reported so far. Many researchers speculate about thepossibility that membrane-bound carotenogenic enzymes would form anenzyme complex. In such a model, the affinity among carotenogenicenzymes would be necessary for efficient carotenogenesis. Based on suchan assumption, this color complementation method is not suitable toclone the enzyme involved in the last step of astaxanthin biosynthesis,namely the one from beta-carotene to astaxanthin, because exogenousenzymes might not have affinity to the Phaffia's carotenogenic enzyme inthe sequential reaction of the carotenogenesis.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably throughout. The terms “nucleic acid” and“polynucleotide” are likewise used interchangeably.

The term “nucleic acid” is intended to include, without limitation, DNA,RNA, cDNA, and mRNA. As used herein, the DNA may be genomic, synthetic,or semi-synthetic. Moreover, the nucleic acids of the present inventioninclude single-stranded and double stranded molecules.

As used herein “derived from” means that the protein, polypeptide,and/or polynucleotide exists naturally in an organism, such as forexample, a P. rhodozyma. However, the polypeptides and polynucleotidesof the present invention may be produced/obtained from any source. Thus,the present invention includes recombinant, synthetic and semi-syntheticproteins, polypeptides, and polynucleotides.

The compositions of the present invention are said to be “isolated,”such as for example “isolated polypeptide,” “isolated polynucleotide,”etc. As used herein, the term “isolated” is intended to mean that thepolypeptide or polynucleotide is purified or, at least partiallypurified as set forth in more detail in the examples.

In this invention, P. rhodozyma ATCC96815 which has been redeposited asa Budapest Treaty deposit at the American Type Culture Collection (ATCC)under accession number 74486 on Feb. 18, 1999 and which is blocked forthe reaction from beta-carotene to astaxanthin was used as atransformation host (Schroeder, W. A. and Johnson, E. A., J. Ind.Microbiol. 14, 502-507, 1995). Transformation of this mutant by thegenomic library prepared from the chromosome of a wild type strain of P.rhodozyma ATCC96594 which has also been redeposited as a Budapest Treatydeposit at the American Type Culture Collection (ATCC) under accessionnumber 74438 on Apr. 8, 1998 was used to isolate a clone which producesastaxanthin. In the present invention, such a genetic fragmentcomplementing the reaction from beta-carotene to astaxanthin in P.rhodozyma was isolated and its nucleotide sequence was determined.

Such a gene/DNA of the present invention can be used for overproductionof astaxanthin through a gene dosage effect using gene amplification orpromoter modification other than complementation of blocked mutation.

In general, a gene consists of several parts which have differentfunctions. In eukaryotes, genes which encode a corresponding protein aretranscribed to premature messenger RNA (pre-mRNA), differing from thegenes for ribosomal RNA (rRNA), small nuclear RNA (snRNA) and transferRNA (tRNA). Although RNA polymerase II (PolII) plays a central role inthis transcription event, PolII cannot solely start a transcriptionwithout a cis element covering an upstream region containing a promoterand an upstream activation sequence (UAS), and a trans-acting proteinfactor. At first, a transcription initiation complex which consists ofseveral basic protein components recognizes the promoter sequence in the5′-adjacent region of the gene to be expressed. In this event, someadditional participants are required if the gene is expressed under somespecific regulation, such as a heat shock response, or adaptation to anutrition starvation, and so on. In such a case, a UAS is required toexist in the 5′-untranslated upstream region around the promotersequence, and certain positive or negative regulator proteins recognizeand bind to the UAS. The strength of the binding of the transcriptioninitiation complex to the promoter sequence is affected by such abinding of the trans-acting factor around the promoter, and this enablesregulation of the transcription activity.

After activation of a transcription initiation complex byphosphorylation, a transcription initiation complex initiatestranscription from the transcription start site. Some parts of thetranscription initiation complex are detached as an elongation complexfrom the promoter region to the 3′ direction of the gene (this step iscalled a “promoter clearance event”) and an elongation complex continuestranscription until it reaches a termination sequence that is located inthe 3′-adjacent downstream region of the gene. Pre-mRNA thus generatedis modified in the nucleus by the addition of a cap structure at the capsite which almost corresponds to the transcription start site, and bythe addition of polyA stretches at the polyA signal which is located atthe 3′-adjacent downstream region. Next, intron structures are removedfrom the coding region and exon parts are combined to yield an openreading frame whose sequence corresponds to the primary amino acidsequence of the corresponding protein. This modification in which amature mRNA is generated is necessary for a stable gene expression. cDNAin general terms corresponds to the DNA sequence which isreverse-transcribed from this mature mRNA sequence. It can besynthesized experimentally by a reverse transcriptase derived fromcertain viral species using a mature mRNA as a template.

In this invention, the mutation point of the P. rhodozyma ATCC96815strain which rendered beta-carotene production to P. rhodozyma wild typestrain was determined. From the sequencing result, it was suggested thatthe base change at the splicing sequence of the eighth intron of the ASTgene caused such a phenotype as specific beta-carotene accumulationthrough the improper splicing of mRNA. RT-PCR analysis detected theimproper spliced product for the AST gene and strongly supported theidentification of the mutation point.

This invention also provides the recombinant AST gene which can beexpressed in different host organisms such as E. coli. In thisinvention, a recombinant AST gene was expressed in E. coli and it wasconfirmed that the AST gene's encoded protein product size correspondedto the deduced molecular weight. Biological production of astaxanthincan be realized by using the novel AST gene and such recombinant DNAtechniques.

According to the present invention, the gene coding for the enzyme whichis involved in the last step of astaxanthin biosynthesis was cloned froma cDNA library of P. rhodozyma, and its nucleotide sequence wasdetermined. Furthermore, a part of the genomic DNA including promoterand terminator were cloned and were used to clone the entire geneincluding the promoter and terminator regions.

An entire gene with its coding region, its intron as well as itsregulation regions such as a promoter and terminator were cloned byscreening a genomic library, which was constructed in a phage or plasmidvector in an appropriate host using a labeled cDNA fragment as ascreening probe. Generally, one of the most common host strains for theconstruction of a genomic library is E. coli. As a vector, a phagevector, such as a lambda phage vector, or a plasmid vector such as a pUCvector can be used. A genomic library constructed in this way, e.g. fromP. rhodozyma DNA can be screened using a labeled DNA fragment with aportion of the gene of interest as a probe. Hybridized plaques orcolonies can then be picked and used for subcloning and/or determinationof the nucleotide sequence.

There are several strategies to enhance the desired enzymatic activityof the protein of interest by using its DNA sequence.

One strategy is to use the gene itself in its native form. The simplestapproach is to amplify the genomic sequence including its regulatorysequences such as the promoter and the terminator. This can be done bycloning the genomic fragment coding for the enzyme of interest into anappropriate vector with a selectable marker which functions in P.rhodoyzma. A drug resistance gene coding for an enzyme that enables thehost to survive in the presence of a toxic antibiotic is often used as aselectable marker. The G418 resistance gene harbored in pGB-Ph9 (Wery etal., Gene, 184, 89-97, 1997) is an example of such a vectorconstruction.

As a vector, two types of vectors can be commonly used. One of thesetypes is an integration vector which does not have an autonomousreplicating sequence. The plasmid pGB-Ph9 is an example of this type ofvector. Because such a vector does not have an autonomous replicatingsequence, the above vector cannot replicate by itself and can be presentonly in an integrated form on the chromosome of the host as a result ofa single-crossing recombination using the homologous sequence between avector and the chromosome. By increasing the concentration of thecorresponding drug in the selection medium, the strain in which theintegrated gene is amplified on the chromosome can only survive. Anothertype of vector is a replicable vector which has an autonomousreplicating sequence. Such a vector can exist in a multicopy state. Inthis type of vector, a nutrition complementation maker also can be usedin the host which has an appropriate auxotrophy marker. The P. rhodozymaATCC24221 strain which requires cytidine for its growth is one exampleof such an auxotroph. By using a CTP synthetase as a donor DNA forATCC2422 1, a host vector system using a nutrition complementation canbe established.

Another strategy to overexpress an enzyme of interest is the placementof the gene of interest under a strong promoter. In such a strategy, thegene of interest must not necessarily be in a multicopy state.Furthermore, a promoter whose promoter activity is induced in anappropriate growth phase and an appropriate timing of cultivation can bealso used. Production of astaxanthin accelerates in the late phase ofgrowth, such as the production phase of a secondary metabolite. Forexample, by placing carotenogenic genes under the control of avegetative promoter, the expression of these genes could be induced inthe exponential growth phase and the production of astaxanthin canbecome tied to the growth of the production strain.

In this invention, the promoter and terminator fragments for the triosephosphate isomerase (TPI) gene was cloned from P. rhodozyma as oneexample of such a constitutive promoter and terminator. Moreover,restoration of astaxanthin production was confirmed in the transformantsin which the AST gene was expressed on a different locus (AMY locus onwhich lies the amylase gene) on the chromosome of beta-caroteneproducing P. rhodozyma ATCC96815 driven by a constitutive promoter andterminator derived from the TPI gene.

Still another strategy to overexpress enzymes of interest is mutation inits regulatory elements. For this purpose, a kind of reporter gene, suchas the beta-galactosidase gene, luciferase gene, a gene coding for agreen fluorescent protein, and the like is inserted between the promoterand the terminator sequence of the gene of interest so that all theparts including promoter, terminator and the reporter gene are fused andfunction together. A transformed P. rhodozyma in which the reporter geneis introduced on the chromosome or on the vector can be mutagenized invivo to induce a mutation within the promoter region of the gene ofinterest. The mutation can be monitored by detecting a change ofactivity coded for by the reporter gene. If the mutation occurs in a ciselement of the gene, the mutation point would be determined by therescue of the mutagenized gene and sequencing. This mutation can then beintroduced to the promoter region on the chromosome by the recombinationbetween the native and the mutated promoter sequence. In the same way, amutation in the gene which codes for a trans-acting factor can be made.

A mutation can be also induced by in vitro mutagenesis of a cis elementin the promoter region. In this approach, a gene cassette, containing areporter gene which is fused to a promoter region derived from a gene ofinterest at its 5′-end and a terminator region from a gene of interestat its 3′-end, is mutagenized and then introduced into P. rhodozyma. Bydetecting the difference of the activity of the reporter gene, aneffective mutation would be screened. Such a mutation can be introducedin the sequence of the native promoter region on the chromosome by thesame method as the case of an in vivo mutation approach.

As donor DNA, a gene coding for an enzyme which catalyzes the reactionfrom beta-carotene to astaxanthin could be introduced. A coding sequencewhich is identical to its native sequence, as well as its allelicvariant, a sequence which has one or more amino acid insertions,deletions, and/or substitutions as long as its corresponding enzyme hasthe same type of enzyme activity, can be used. Such a vector can then beintroduced into P. rhodozyma by transformation and the transformants canbe selected by spreading the transformed cells on an appropriateselection medium such as YPD agar medium containing geneticin in thecase of pGB-Ph9 or a minimal agar medium omitting cytidine in the caseof using auxotroph ATCC24221 as a recipient.

Such a genetically engineered P. rhodozyma can be cultivated in anappropriate medium and evaluated for its productivity of astaxanthin. Ahyper producer of astaxanthin thus selected would be confirmed in viewof the relationship between its productivity and the level of gene orprotein expression which is introduced by such a genetic engineeringmethod.

The following examples are provided to further illustrate methods ofpreparation of the enzyme of the present invention, as well as certainphysical properties and uses thereof. These examples are illustrativeonly and are not intended to limit the scope of the invention in anyway.

EXAMPLES

The following materials and methods were employed in the specificExamples described below:

Strains:

P. rhodozyma ATCC96594 (This strain has been redeposited on Apr. 8, 1998as a Budapest Treaty deposit under accession No. 74438)

P. rhodozyma ATCC96815 (This strain has been redeposited on Feb. 18,1999 as a Budapest Treaty deposit under accession No.74486)

E. coli DH5alpha F⁻, phi80d, lacZdeltaM15, delta(lacZYA-argF)U169, hsd(r_(K) ⁻, m_(K) ⁺), recA1, endA1, deoR, thi-1, supE44, gyrA96, relA1(Toyobo, Osaka, Japan)

E. coli XL1-Blue MRF′: delta(mcrA)183, delta(mcrCB-hsdSMR-mrr)173,endA1, supE44, thi-1, recA1, gyrA96, relAn1, lac(FproAB,lacI^(q)ZdeltaM15, Tn10 (tet^(r))) (Stratagene, La Jolla, USA)

E. coli SOLR: e14⁻(mcrA), delta(mcrCB-hsdSMR-mrr)171, sbcC, recB, recJ,umuC::Tn5(kan^(r)), uvrC, lac, gyrA96, relA1, thi-1, endA1, lambda^(R),(F′ proAB, lacI^(q)ZdeltaM15) Su⁻(nonsuppressing) (Stratagene)

E. coli TOP10:F⁻, mcrA, delta(mrr-hsdRMS-mcrBC), phi80, delta(lacZ M15),delta(lacX74), recA1, deoR, araD139, (ara-leu)7697, galU, galK,rpsL(Str^(r)), endA1, nupG (Invitrogen, NV Leek, Netherlands)

E. coli BL21 (DE3) (pLysS): dcm⁻, ompTrB⁻mB⁻, Ion⁻ lambda(DE3), pLysS(Stratagene)

Vectors:

pUC19 (Takara Shuzo, Otsu, Japan)

lambdaZAPII (Stratagene)

pCR2.1-TOPO (Invitrogen)

pET11c (Stratagene)

Media

The P. rhodozyma strain is maintained routinely in YPD medium (DIFCO,Detroit, USA). The E. coli strain is maintained in LB medium (10 gBacto-trypton, 5 g yeast extract (DIFCO) and 5 g NaCl per liter). Whenan agar medium was prepared, 1.5 % of agar (WAKO, Osaka, Japan) wassupplemented.

Methods

General molecular biology methods were done according to those describedin Molecular Cloning: a Laboratory Manual, 2nd Edition (Cold SpringHarbor Laboratory Press, 1989). Restriction enzymes and T4 DNA ligasewere purchased from Takara Shuzo.

Isolation of chromosomal DNA from P. rhodozyma was performed using aQIAGEN Genomic Kit (QIAGEN, Hilden, Germany) following the protocolsupplied by the manufacturer. A mini-prep of plasmid DNA fromtransformed E. coli was performed with the Automatic DNA isolationsystem (PI-50, Kurabo, Co. Ltd., Osaka, Japan). A midi-prep of plasmidDNA from an E. coli transformant was performed using a QIAGEN column(QIAGEN). A DNA fragment was isolated and purified from agarose usingQIAquick or QIAEX II (QIAGEN).

Fluorescent DNA primers for DNA sequencing were purchased fromPharmacia. DNA sequencing was performed with the automated fluorescentDNA sequencer (ALFred, Pharmacia, Uppsala, Sweden).

Competent cells of DH5alpha were purchased from Toyobo.

The apparatus and reagent for biolistic transformation of P. rhodozymawere purchased from Nippon Bio-Rad Laboratories (Tokyo, Japan).

Example 1 Isolation of Genomic DNA from P. rhodozyma

To isolate a genomic DNA from P. rhodozyma, ATCC96594 the QIAGEN genomickit was used according to the method specified by the manufacturer.

At first, cells of P. rhodozyma ATCC96594 from 100 ml of overnightculture in YPD medium were harvested by centrifugation (1500×g for 10min.) and washed once with TE buffer (10 mM Tris/HCl (pH 8.0) containing1 mM EDTA). After suspending in 8 ml of Y1 buffer of the QIAGEN genomickit, lyticase (SIGMA, St. Louis, USA) was added at a concentration of 2mg/ml to disrupt the cells by enzymatic degradation. The reactionmixture was incubated for 90 minutes at 30° C. and then proceeded to thenext extraction step. Finally, 20 μg of genomic DNA was obtained.

Example 2 Construction of a Genomic Library from P. rhodozyma ATCC 96594

As described in the section “detailed description of the invention”, aplasmid harboring a drug resistant marker cassette was constructed byinserting a G418 resistant structure gene between the promoter andterminator region of the gene of the glyceraldehyde-3-phosphatedehydrogenase (GAP) and ligating this cassette into the KpnI- andHindIII- digested pUC19. This plasmid was named pUC-G418 and usedfurther on. Then, a ClaI linker was ligated into the unique EcoRI siteof the pUC-G418 vector and the resultant plasmid pUC-G418C1512 wasobtained and used as a vector backbone in the construction of thePhaffia's genomic library.

Then, 10 μg of chromosomal DNA prepared from P. rhodozyma ATCC96594 asdescribed above in Example 1 was digested partially with 1.6 units ofHpaII for 45 minutes at 37° C. and was subjected to agarose gelelectrophoresis. After staining by ethidium bromide, partially digestedDNA species (i.e., fragments) from 4 to 10 kb were recovered byelectroelution using a dialysis membrane. After ethanol precipitation ofrecovered HpaII fragments, 1.215 μg of DNA was obtained. Next, 3 μg ofpG418Cl512 was digested by 10 units of ClaI for an hour at 37° C. andprecipitated with ethanol. ClaI-digested pG418C1512 was thendephosphorylated using calf intestine alkaline phosphatase.ClaI-digested and dephosphorylated pG418Cl512 vector was then subjectedto agarose gel electrophoresis and the DNA fragment was recovered usingthe QIAquick protocol according to the instructions of the manufacturer.Finally, 2.62 pg of ClaI-digested and dephosphorylated pG418Cl512 wasobtained.

2.62 μg of ClaI-digested and dephosphorylated pG418C1512 was ligated to1.22 μg of HpaII-partially digested Phaffia's genomic DNA over night at16° C. and the resultant ligation solution was used as donor DNA for thetransformation of an E. coli DH5alpha strain. The total ligation mixture(270 μl) was transferred to 1 ml of competent cells of DH5alpha(Toyobo). After a heat shock treatment at 42° C. for 45 secondssucceeded by maintenance on ice for 30 minutes, the transformed cellswere placed on ice for 2 minutes and then incubated at 37° C. for anhour with the addition of 5 ml of SOC medium containing:

0.5% yeast extract (DIFCO)

2% trypton (DIFCO)

10 mM NaCl

2.5 mM KCl

10 mM MgCl₂

20 mM MgSO₄

20 mM glucose.

The incubated cells were transferred into 100 ml of LB medium containing100 microgram/ml of ampicillin. Cultivation was continued overnight at37° C. and then the cells were harvested for plasmid midi-preparation.

A plasmid library was prepared from harvested cells using QIAGENmidi-prep columns according to the method supplied by the manufacturer.Finally, 0.3 mg/ml of Phaffia's genomic library was obtained in a totalvolume of 5 ml and used as a genomic library.

Example 3 Transformation of P. rhodozyma ATCC96815 with a BiolisticMethod

Transformation was done according to the method described in Methods inMolecular Biology (Johnston et al., 53; 147-153, 1996). As a hoststrain, P. rodozyma ATCC96815 was cultured in YPD medium to thestationary phase. After centrifugation of the broth, cells wereconcentrated 10-fold with sterilized water and 200 μl of the cellsuspension was spread on YPD medium containing 100 microgram/ml ofgeneticin, and 0.75 M of mannitol and sorbitol. Five micrograms of agenomic library, prepared as described in Example 2, was coated on 1.5mg of 0.9 μm gold particle, and used as donor DNA for the biolistictransformation. Approximately 20,000 geneticin-resistant clones wereyielded (300 to 500 colonies per plate) after one week of incubation at20° C. Although most of the transformants showed a yellow color (as thehost strain, ATCC96815 did), three colonies pigmented red and were usedfurther on.

Example 4 Analysis of Carotenoid Obtained From Red-pigmentedTransformants

Red-pigmented transformants obtained from P. rhodozyma ATCC96815(Example 3) were cultivated in 10 ml of YPD medium at 20° C. in testtubes. Then, cells were harvested from 0.5 ml of broth and used for theextraction of carotenoids from cells. The carotenoid content of P.rhodozyma was measured by HPLC after extraction of the carotenoids fromcells of P. rhodozyma by disruption with glass beads as described. Afterextraction, disrupted cells were then collected by centrifugation andthe resultant supernatant was analyzed for carotenoid content by HPLC.

HPLC column; Chrompack Lichrosorb si-60 (4.6 mm, 250 mm)

Temperature; room temperature

Eluent; acetone/hexane (18/82) add 1 ml/L of water to elluent

Injection volume; 10 μl

Flow Rate; 2.0 ml/minute

Detection; UV at 450 nm

A sample of beta-carotene was purchased from SIGMA and astaxanthin wasobtained from Hoffman La-Roche (Basel, Switzerland).

As a result of HPLC analysis, it was confirmed that all three redtransformants produced astaxanthin specifically though the host strain,ATCC96815 produced only beta-carotene.

Example 5 Plasmid Rescue From the Chromosome of Red Transformants (ofExample 3) Which Produced Astaxanthin

Chromosomal DNA was prepared from all the astaxanthin-producingtransformants. For this purpose, the QIAGEN genomic kit was usedaccording to the method specified by the manufacturer, as described inExample 1. 5 μg of chromosomal DNA, thus prepared, was digested byHindIII and then purified according the QIAquick protocol. E. coliDH5alpha competent cells were transformed by the ligated DNA solutionsand then spread on LB agar medium containing 100 μg/ml of ampicillin.All of the transformants had the same insert fragments in theirplasmids, judging from sequence analysis of the plasmids. This indicatedthat three independent red transformants derived from P. rhodozymaATCC96815 were yielded by the same type of recombination event betweenthe donor DNA of the genomic library and chromosomal DNA. One of theplasmids thus rescued was named pR2-4 and used further on.

Example 6 Screening of the Original Genomic Library By Using pR2-4 as aHybridization Probe

Because the rescued fragment in pR2-4 may have mutations depending onthe direction of the recombination event yielding red transformants ofP. rhodozyma, screening of the original genomic library was done byusing pR2-4 as a hybridization probe.

For this purpose, twenty thousand E. coli transformants of the originalgenomic library, as described in Example 2 were transferred to nylonmembrane filters (Hybond-N+, Amersham, Buckinghamshire, UK) andsubjected to colony hybridization. Three transformants which harboredthe same nucleotide sequence in their insert as that of pR2-4 wereisolated. The isolated plasmids from these transformants were named pR3,pR5.1 and pR16.

Next, P. rhodozyma ATCC96815 was transformed by pR3, pR5.1 and pR16. Allthe transformants colored red. This result suggests that the isolatedplasmids might contain the gene encoding an enzyme involved in thereaction of beta-carotene to astaxanthin in P. rhodozyma. We designatedthis gene as AST gene. Among these plasmids, pR16 was used further on.

Example 7 Isolation of mRNA From P. rhodozyma for cDNA Analysis

To analyze the pattern of transcripts from P. rhodozyma, total RNA wasisolated from P. rhodozyma ATCC96594 and ATCC96815 by phenol extractionby combination of the cell disruption with glass beads and purified mRNAusing an mRNA separation kit (Clontech, Palo Alto, USA).

At first, cells of ATCC96594 and ATCC96815 strains from 10 ml of atwo-day-culture in YPD medium were harvested by centrifugation (1500×gfor 10 min.) and washed once with extraction buffer (100 mM Tris/HCl (pH7.5) containing 0.1 M LiCl and 0.1 mM EDTA). After filling up to 5.0 mlof cell suspension with the same extraction buffer in 50 ml disposablecentrifuge tube (IWAKI Glass, Tokyo, Japan), 1.5 ml of isogen-LS (Nippongene, Toyama, Japan) and 10 grams of glass beads were added. Centrifugetubes which contained the cell suspension with isogen-LS and glass beadswere shaken with a horizontal table top shaker for an hour. In thisstep, 300 μg of total RNA was recovered.

Then, mRNA was purified using an mRNA separation kit (Clontech). On 8.0μg of mRNA from P. rhodozyma ATCC96594 and ATCC96815 strains wereobtained.

To synthesize cDNA, we used the SMART cDNA construction kit (Clontech)according to the method specified by the manufacturer. We applied 2 μgof purified mRNA for a first strand synthesis followed by PCRamplification and obtained 1 mg of cDNA.

Example 8 Subcloning of pR16 and Functional Analysis of its InsertFragment

The restriction map of pR16 is depicted in FIG. 12. Each EcoRI fragmentwhose length was 0.7 and 2.7 kb, was subcloned into pUC-G418 and namedpRS913 and pRLR913, respectively.

Then, the astaxanthin-producing P. rhodozyma ATCC96594 strain wastransformed with pRS913. As a result of this transformation study,yellow transformants were yielded. This suggested that 0.7 kb EcoRIfragment might contain a truncated AST gene and the transformation via asingle-cross recombination between a 0.7 kb EcoRI fragment and itshomologous sequence on the choromosome of P. rhodozyma would result in agene disruption of the AST gene on the choromosome of P. rhodozyma.

Next, the beta-carotene-producing ATCC96815 strain was transformed withpRLR913 and red transformants were yielded. This suggested that themutation point of strain ATCC96815 which led the astaxanthin-producingwild type strain to produce beta-carotene would lie in the 2.7 kb EcoRIfragment originally adjacent to the 0.7 kb EcoRI fragment in pR16.

Two hundred μg of cDNA prepared in Example 7 was subjected to agarosegel electrophoresis for virtual Northern analysis. In the case of thecDNAs prepared from ATCC96594 and 96815, two bands which, namely at 3.2and 2.0 kb were hybridized in both cases by using the 2.7 kb EcoRIfragment of pRLR913 as a hybridization probe. This suggested that theast mutation of ATCC96815 would be a point mutation which did notreflect a change in the length of mRNA such as a missense mutation.

In the case of using the 0.7 kb EcoRI fragment of pRS913 as ahybridization probe, a band of 2.0 kb was hybridized. From this study,it seemed that the AST gene might give a 2.0 kb transcript in P.rhodozyma.

Example 9 Cloning of the cDNA of the ast Gene

To clone the cDNA for the AST gene from P. rhodozyma, we constructedcDNA library from P. rhodozyma ATCC96594. Total RNA was isolated byphenol extraction by combination of the cell disruption with glass beadsas described in Example 7.

At first, cells of the ATCC96594 strain from 50 ml of a two-day-culturein YPD medium were harvested by centrifugation (1500×g for 10 min.) andwashed once with extraction buffer (100 mM Tris/HCl (pH 7.5) containing0.1 LiCl and 0.1 mM EDTA). After filling up to 5.0 ml of cell suspensionwith the same extraction buffer in 50 ml disposable centrifuge tubes(IWAKI Glass), 1.5 ml of isogen-LS (Nippon gene) and 10 grams of glassbeads were added. Centrifuge tubes which contained cell suspension withisogen-LS and glass beads were shaken with a horizontal table top shakerfor an hour. In this step, 1.8 mg of total RNA was recovered.

Then, mRNA was purified using the PolyATtract mRNA isolation system(Promega corp., Madison, USA) according to the method specified by themanufacturer. Finally, we obtained 8.0 μg of mRNA from the P. rhodozymaATCC96594 strain.

To construct a cDNA library, 8.0 μg of the purified mRNA was used in theCOPY kit (Invitrogen, Carlsbad, USA) with the protocol specified by themanufacturer. After ligation of an EcoRI adaptor (Stratagene),synthesized cDNA was subjected to agarose gel electrophoresis. After theexcision of the agarose gel piece which covered the length of cDNAs from1.9 to 2.3 kb, the collected cDNA species were purified by QIAEX II(QIAGEN). This size-fractionated cDNA was ligated to EcoRI-digested anddephosphorylated lambdaZAPII (Stratagene). The over-night ligationmixture was in vitro packaged with Gigapack III gold extract(Stratagene) and used to infect an E. coli XL1-Blue MRF′ strain.

Conventional plaque screening was performed against 6000 plaques using2.7 and 0.7 kb EcoRI fragment as described in Example 8 as hybridizationprobes. One plaque hybridized strongly to these probes, and was pickedup with a sterilized toothpick and the eluted phage particle were usedfor in? vivo excision, according to the method specified by themanufacturer. Finally, infected transformants of E. coli SOLR cellswhich showed resistance against ampicillin were isolated. Aftersequencing of the isolated plasmids obtained from these transformants,it turned out that these plasmids contained the same fragment as a partof the sequence of pR6 which was described in Example 6.

The entire sequence of the cDNA of the AST gene was determined and isshown as SEQ ID NO: 2 and its deduced amino acid sequence as SEQ ID NO:1.

Example 10 Expression of AST Gene in E. coli

To confirm that the ORF for the AST gene actually encodes a protein, anexpression study of the AST gene was performed in an E. coli expressionsystem. At first, a 6×histidine (His) tag was added to the carboxylterminal end of the AST product in order to make it easy to purify. PCRprimers whose sequences are listed in TABLE 1 were synthesized.

Table 1

PCR primers for cloning 3′ portion of AST gene to which a 6×His tag isadded

ast13:

GTTCAAAGTTCATTTATGGA (sense primer) (SEQ ID NO: 4)

ast14:

GGATCCTCAGTGGTGGTGGTGGTGGTGTTCGACCGGCTTGACCTGCA (antisense primer) (SEQID NO: 5)

Next, 1.5 kb of NdeI/EcoRI fragment of pAST1207 and 0.3 kb ofEcoRI/BamHI fragment of pAST 114 were ligated into pET11c which wasdigested by NdeI and BamHI and ligated DNA was transformed into E. coliJM109 strain. Six independent ampicillin resistant clones were examinedby restriction analysis and it was found that 5 of 6 clones had thecorrect structure of the recombinant expression plasmid containing ASTgene. One of these clones was selected for further study (pAST 120) andthen transformed into E. coli BL21 (DE3) (pLysS) strain. It was revealedthat all of the ampicillin resistant clones which were examined byrestriction analysis possessed pAST 120 properly.

Next, an expression study was performed by addition of 1 mM IPTG to E.coli BL21 (DE3) (pLysS) (pAST120) growing culture when the opticaldensity (OD) at 600 nm reached 0.8. After continuation of cultivation at37° C. for 4 hours, cells were harvested by centrifugation and lysed byboiling in SDS sample buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4%SDS, 0.005% bromophenol blue, 5% mercaptoethanol). The lysate was thensubjected to SDS-polyacrylamide gel electrophoresis (PAGE). Expressedprotein was not observed after staining by coomassie brilliant blue(Rapid stain CBB kit, nacalai tesque, Kyoto, JAPAN) (data not shown).

In general, it is reported that some modifications of amino acidsequence at the amino terminal region of the P450 protein is required toexpress P450 protein in an E. coli expression system. In fact, the ASTgene which had an intact sequence was not expressed in E. coli (data notshown) and it was found that some modifications the amino terminalsequence was necessary in the case of the AST gene as well as other P450enzymes. As a next strategy for expression of a recombinant AST gene,the construction, in which 6×His tag sequence was added at the aminoterminal end of the AST protein on the deletion of the hydrophobicanchor sequences which were located at the amino terminal end of the ASTgene, was made.

In order to add a 6×His tag sequence at the amino terminal end of theAST protein, anchor sequences were deleted at the amino terminal end,and the following PCR primers were synthesized (Table 2) and used forPCR cloning.

Table 2

PCR primers for cloning the AST gene lacking an anchor sequence at its5′ portion to which 6×His tag is added

ast32:

CATATGCACCACCACCACCACCACCTGTATAACCTTCAGGGGCCC (sense primer for cloningof 5′ end of AST gene) (SEQ ID NO: 6)

ast2:

GTAACAACACCATCTCCGGT (antisense primer for cloning of 5′ end of ASTgene) (SEQ ID NO: 7)

ast13:

GTTCAAAGTTCATTTATGGA (sense primer for cloning of 3′ end of AST gene)(SEQ ID NO: 4)

ast33:

GGATCCTCAACTCATTCGACCGGCTT (antisense primer for cloning of 3′ end ofAST gene) (SEQ ID NO: 8)

The PCR conditions were as follows: 25 cycles of 15 seconds at 94° C.,30 seconds at 55° C. and 30 seconds at 72° C. The plasmid, pAST1207 wasused as PCR template. PCR fragments which had the desired length werecloned into pCR2.1-TOPO (Invitrogen) and 6 independent clones which hadexpected inserts were examined for their insert sequence. As a result,two of the clones had the exact insert sequence, and one clone wasselected and used for further study (pAST228 for 3′ end of AST gene andpAST302#3202 for 5′ end of AST gene, respectively). A 0.2 kb NdeI/SphIfragment from pAST302#3202, 1.5 kb SphI/EcoRI fragment from pAST1207 and0.05 kb KpnI/BamHI fragment from pAST228 were ligated into pET11cdigested by NdeI and BamHI and the ligated mixture was transformed intoE. coli DH5alpha.

As a result of restriction analysis for 6 independent clones, it wasfound that all the clones had the correct structure harboring the ASTgene for its expression. One clone was selected and used for furtherstudy (pAST315). Next, pAST315 was transferred into expression host E.coli BL21 (DE3) (pLysS). It was confirmed that all the 6 transformantshad pAST315 correctly as a result of restriction analysis.

Next, an expression study was performed by addition of 1.5 mM IPTG to E.coli BL21 (DE3) (pLysS) (pAST315) growing culture when the opticaldensity (OD) at 600 nm reached 0.93. After continuation of cultivationat 37° C. for 4 hours, cells were harvested by centrifugation and lysedby boiling in SDS sample buffer. The lysate was then subjected to PAGE.An expressed protein whose molecular weight corresponded well with itsdeduced amino acid sequence (approximate 60 kDa) was observed afterstaining by coomassie brilliant blue (FIG. 13). From this result, it wasconfirmed that the AST gene encodes a protein expected from its deducedopen reading frame.

Example 11 In Vitro Characterization of the AST Gene Product

For the enzymatic characterization of the AST gene product, a standardassay which is used for the characterization of P450 enzymes can beapplied. For this purpose, it is necessary that the reaction mixturecontains a reconstituted membrane. As a reconstituted membrane, naturalisolates such as mitochondrial membranes or microsomes and artificialmembranes are often used. It is necessary that an electron transferbetween an electron acceptor and a receptor can occur. As an electrondonor, cytochrome P450 reductase is often added to the reaction mixture.As an electron acceptor, oxygen molecules are involved. Under thepresence of an electron source, such as reduced NADPH+, beta-carotene,which is a substrate of astaxanthin synthase can be converted toastaxanthin. Produced astaxanthin can be assayed qualitatively andquantitatively with HPLC analysis.

Example 12 Cloning of a Genomic Fragment Containing the AST Gene

To determine the genomic sequence containing the AST gene, a sequencingexperiment was performed using a primer-walking procedure. Sequencinganalysis of pRS913 showed that pRS913 did not contain the 3′ end of theAST gene. To obtain the 3′-adjacent genomic fragment to the AST gene, agenome-walking experiment was performed. To do this, a universal genomewalker kit (Clontech) was exploited according to the method specified bythe manufacturer. As a template of PCR, chromosomal DNA prepared inExample 1 was used. A gene specific primer, ast15 whose sequence was aslisted in TABLE 3 was synthesized and used as a PCR primer.

Table 3

Sequence of primer used for genome walking of the AST gene

ast15:

TAGAGAGAAGGAGGGGTACCAGATGC (SEQ ID NO. 9)

PCR fragments an which had appropriate length (smaller than 1 kb) wereobtained from an EcoRV and StuI library, purified and cloned intopCR2.1-TOPO (Invitrogen). As a result of sequencing, it was found thatboth fragments contained the genomic fragment encoding the AST gene.Based on the sequence which was located at 200 bp from the polyA sitefor the AST gene, PCR primer was designed as listed in TABLE 4.

Table 4

Sequence of primer for cloning of 3′-adjacent fragment to the AST gene

ast18:

CCCCGGATTGTGGAGAAACT (SEQ ID NO: 10)

By using ast15 and ast18 primers as PCR primers and chromosomal DNAprepared in Example 1 as PCR template, PCR was conducted. Proof-readingpolymerase (HF polymerase, Clontech) ensured the amplification of a PCRfragment which had the exact sequence. PCR condition was as follows; 25cycles of 15 seconds at 94° C., 30 seconds at 55° C. and 30 seconds at72° C. Six independent clones which had 400 bp inserts showed theidentical sequence.

By combining the sequences from pRS913 and pRL913, a 3.9 kb sequencecontaining the AST gene having a 474 bp promoter region and a 269 bpterminator region was determined (SEQ ID NO. 3). As a result, the ASTgene showed intron-rich structure (17 introns).

Example 13

Determination of Mutation Point in Beta-carotene Producing Strain, P.rhodozyma ATCC96815

To confirm the fact that beta-carotene production by the P. rhodozymaATCC96815 strain was caused from the mutation within the AST gene, agenomic sequence containing the AST gene obtained from ATCC96815 and itsparent strain P. rhodozyma ATCC24230 were determined. To do this, PCRprimers whose sequences were as listed in TABLE 5 were synthesized andused for PCR cloning.

Table 5

PCR Primers for Cloning the Entire Genomic ast Gene;

ast21:

ATGTTCATCTTGGTCTTGCT (sense primer) (SEQ ID NO: 11)

ast4:

ACGTAGAAGTCATAGCGCCT (antisense primer) (SEQ ID NO: 12)

By using HF polymerase (Clontech) as PCR polymerase and chromosomal DNAprepared from strains, ATCC96815 and ATCC24230 by the same protocol asExample 1 as PCR template, PCR was performed under the condition asfollows; 25 cycles of 15 seconds at 94° C., 30 seconds at 55° C. and 4minutes at 72° C. PCR fragments obtained whose length were approximately3.5 kb were cloned into pCR2.1-TOPO and sequenced for their entiresequences by the primer walking procedure. Between the sequence for P.rhodozyma ATCC96594 strain and ATCC24230 strain, 7 base changes werefound. Four base changes were found in its exon sequence but those didnot give any amino acid changes. Three base changes were found in itsintron structure. In comparison between beta-carotene producing strainATCC96815 and its parent strain, ATCC24230, one base change which waslocated at the 5′-splicing sequence (GTAAGT>GTAAAT) within the eighthintron was found. This might indicate that the mutation which conferredthe phenotype of beta-carotene accumulation on astaxanthin-producing P.rhodozyma was caused by improper splicing of the mRNA for the AST gene.

To confirm this assumption, RT-PCR was performed using cDNA preparedfrom P. rhodozyma ATCC96815 as PCR template. mRNA was isolated from P.rhodozyma ATCC96815 by the same protocol as Example 9 and used for thesynthesis of cDNA. To obtain cDNA from this mRNA prepared from ATCC96815by the PCR method, a SMART PCR cDNA library construction kit (Clontech)was exploited according to the method specified by supplier. Thefollowing primers whose sequence are as listed in TABLE 6 and whichcovered the eighth intron were synthesized and used for PCR primers.

Table 6

PCR primers for RT-PCR to detect the improper splicing product for theAST gene

ast7:

TTTGACTCAAGGATTAGCAG (sense primer) (SEQ ID NO: 13)

ast26:

TGTCTTCTGAGAGTCGGTGA (antisense primer) (SEQ ID NO: 14)

RT-PCR conditions were as follows: 25 cycles of 15 seconds at 94° C., 30seconds at 55° C. and 30 seconds at 72° C. As a result of PCR, 300 bp ofPCR products were amplified and cloned into pCR2.1-TOPO. Two independentclones which had 300 bp inserts were sequenced. As a result, it wasconfirmed that improper splicing products for the AST gene weresynthesized in the P. rhodozyma ATCC96815 strain. Improper splicing inthe eighth intron of the AST gene might cause the production of shortertruncated AST proteins than the AST protein spliced properly because astop codon lies in the eighth intron. This result indicated that themutation point lies in the AST gene which failed in the proper splicing.

Example 14 Expression of the AST Gene in a Beta-carotene-producingPhaffia rhodozyma

To confirm that the AST gene encoded the enzyme which was involved inthe conversion of beta-carotene to astaxanthin, the AST gene was clonedinto a beta-carotene-producing strain. To exclude the possibility ofrecombination at a native locus of the AST gene on the chromosome, anexpression plasmid for the AST gene on the AMY locus of Phaffiarhodozyma's chromosome was constructed. To do this, cloning of somegenetic elements from Phaffia rhodozyma was required.

1) Cloning of the Constitutive Promoter and Terminator from Phaffiarhodozyma

To clone a constitutive promoter and terminator from Phaffia rhodozyma,a degenerate PCR method was exploited. Among the genes which are oftenused as a constitutive promoter and terminator in yeast genetics, theTPI gene which encode triose phosphate isomerase was cloned. Among theconserved amino acid sequence registered in Blocks database(http://www.blocks.fhcrc.org/), two motif sequences (Arg-Thr-Phe-Phe-Val-Gly-Gly-Asn and Asp-Val-Asp-Gly-Phe-Leu-Val-Gly-Gly-Ala) wereselected and their degenerate primers were synthesized as follows.

Table 7

Degenerate PCR primers for cloning of the TPI gene from P. rhodozyma

tp1:

MGNACNTTYTTYGTNGGNGGNAAY (sense primer) (SEQ ID NO: 15)

tp6:

GCNCCNCCNACNARRAANCCRTCNACRTC (antisense primer) (SEQ ID NO: 16)

(M=A or C; N=A, C, G or T; Y=C or T; R=A or G)

PCR conditions were as follows: 25 cycles of 15 seconds at 94° C., 30seconds at 46° C. and 15 seconds at 72° C. ExTaq polymerase (TakaraShuzo) was used as the PCR polymerase. As a PCR template, a cDNA poolwas prepared from mRNA isolated from P. rhodozyma ATCC96594 using aSMART PCR cDNA library construction kit (Clontech). A 0.7 kb PCRfragment was purified and cloned into pCR2.1-TOPO. Six independentclones had inserts having the desired length, judging from restrictionanalysis. Two of these clones were sequenced and it was confirmed thatboth of them had an insert sequence which had striking homology to knownTPI genes from various organisms. One of these clones was selected forfurther study (pTPI923).

Next, based on the insert sequence of pTPI923, several PCR primers whosesequences are listed in TABLE 8 were synthesized for genome walking toclone the promoter and terminator of the TPI gene. For this experiment,an universal genome walker kit (Clontech) was exploited according to themethod specified by the manufacturer.

Table 8

PCR primers for genome walking to clone the TPI promoter and terminator

tp9:

GCTTACCTCGCTTCCAACGTTTCCCAG (terminator cloning, primary) (SEQ ID NO:17)

tp10:

GGATCTGTCTCTGCCTCCAACTGCAAG (terminator cloning, nested) (SEQ ID NO: 18)

tp11:

GGGTCAATGTCGGCAGCGAGAAGCCCA (promoter cloning, primary) (SEQ ID NO: 19)

tp12:

ATGTACTCGGTAGCACTGATCAAGTAG (promoter cloning, nested) (SEQ ID NO: 20)

PCR conditions were as follows: 7 cycles of 4 seconds at 94° C. and 3minutes at 74° C., followed by 32 cycles of 4 seconds at 94° C. and 3minutes at 69° C. and succeeded to extension at 69° C. for 4 minutes.KOD polymerase (TOYOBO) was used as the PCR polymerase. Chromosomal DNAprepared from P. rhodozyma ATCC96594 was used as a PCR template. As aresult, candidate for the terminator region was obtained from the EcoRVand StuI library. Sequencing analysis for these candidates revealed thatboth clones had the downstream sequence for the TPI gene containing thededuced 3′ end of the TPI structural gene and terminator region. In caseof cloning for the promoter region′ candidates which were obtained fromthe EcoRV library contained the deduced 5′ end of the TPI structuralgene and promoter region.

Then, PCR primers whose sequences are listed in TABLE 9 were synthesizedfor the construction of promoter cassette and terminator cassettederived from TPI gene.

Table 9

PCR primers to construct the TPI promoter and the TPI terminatorcassette

tp13:

GCGGCCGCATCCGTCTCGCCATCAGTCT (sense primer for promoter cassette) (SEQID NO: 21)

tp14:

CCTGCAGGTCTAGAGATGAATAAATATAAAGAGT (antisense primer for promotercassette) (SEQ ID NO: 22)

tp15:

CCTGCAGGTAAATATATCCAGGGATTAACCCCTA (sense primer for terminatorcassette) (SEQ ID NO: 23)

tp16:

GGTACCCGTGCGCAGTCGACCGAGACAT (antisense primer for terminator cassette)(SEQ ID NO: 24)

PCR condition were as follows: 25 cycles of 15 seconds at 94° C., 30seconds at 55° C. and 30 seconds at 72° C. HF polymerase (Clontech) wasused as the PCR polymerase and yielded PCR fragments which were clonedinto pCR2.1-TOPO. As a result of restriction and sequencing analysis, itwas found that clones which had identical sequences were obtained. Eachclone was selected for further study (pTPIP1104 for promoter cassetteand pTPIT1104 for terminator cassette, respectively).

2) Cloning of Partial Amylase Gene from Phaffia rhodozyma

To locate and express a foreign gene on the chromosome of P. rhodozyma,the amylase gene was cloned from P. rhodozyma. In case that expressionvector on which the foreign gene would be cloned could contain anhomologous genetic fragment to the chromosomal sequence of P. rhodozyma,such as an amylase gene, an expression vector can be integrated on thehomologous region on the chromosome of P. rhodozyma after the singlecross recombination.

Eleven amino acid sequences encoding amylase from various organisms wereselected from the Entrez database (http://www.ncbi.nlm.nih.gov/Entrez/)and used for amino acid alignment by clustal W (Thompson, J. D.,Higgins, D. G. and Gibson, T. J., Nucleic Acids Research, 22: 4673-4680,1994). The eleven organisms whose amylase sequences were registered onthe database are as listed in TABLE 10.

Table 10

Various amylase genes which were registered on the database for clustalW analysis

Aspergillus niger var. awamori amyA gene (accession number X52755)

Aspergillus niger var. awamori amyB gene (accession number X52756)

Aspergillus kawachii acid-stable alpha-amylase gene (accession numberAB008370)

Aspergillus oryzae amyl gene (accession number X12725)

Aspergillus shirousamii alpha-amylase gene (accession number P30292)

Cryptococcus species alpha-amylase gene (accession number D83541)

Lipomyces kononenkoae subsp. spencermartinsiae alpha-amylase gene(accession number U30376)

Debaryomyces occidentalis amy1 gene (accession number X 16040)

Saccharomycopsis fibuligera ALP1 gene (accession number X05791)

Schizosaccharomyces pombe alpha-amylase gene (accession number Z64354)

Two conserved amino acid sequences (Asp-Tyr-Ile-Gln-Gly-Met-Gly-Phe-Asp/Thr-Ala-Ile-Trp andAsp-Gly-Ile-Pro-Ile-Ile-Tyr-Tyr-Gly-Thr-Glu-Gln) for amylase wereselected to clone the amylase gene from P. rhodozyma by a degenerate PCRmethod. Then, PCR primers whose sequences are listed in TABLE 11 weresynthesized for the cloning of the AMY gene from P. rhodozyma.

Table 11

Degenerate PCR primers for cloning of amylase (AMY) gene from P.rhodozyma

amy1:

GAYTAYATHCARGGNATGGGNTTYRMNGCNATHTG (sense primer) (SEQ ID NO: 25)

amy2:

TGYTCNGTNCCRTARTADATDATNGGDATNCCRTC (antisense primer) (SEQ ID NO: 26)

(Y=C or T; H=A, C or T; R=A or G; N=A, C, G or T; M=A or C; D=A, G or T)

PCR conditions were as follows: 25 cycles of 15 seconds at 94° C., 30seconds at 50° C. and 2 minutes at 72° C. ExTaq polymerase (TakaraShuzo) was used as PCR polymerase. As a PCR template, chromosomal DNAprepared in Example 1 and the cDNA pool prepared from mRNA isolated fromP. rhodozyma ATCC96594 using the SMART PCR cDNA library construction kit(Clontech) were used. 1.7 kb and 0.9 kb PCR fragments were yielded whenchromosome and cDNA were used as PCR template, respectively. Bothfragments were purified and cloned into pCR2.1-TOPO. Six independentclones had inserts having the desired length, judging from restrictionanalysis. Two of these clones were sequenced and it was confirmed thatboth of them had insert sequence which had striking homology to knownamylase genes from various organisms. One of these clones whichcontained a chromosomal AMY fragment was selected for further study(pAMY216). To construct a partial amylase cassette, two PCR primerswhose sequences are listed in TABLE 12 were synthesized based on theinternal sequence of the insert fragment of pAMY216.

Table 12

PCR primers to construct a partial AMY cassette

amy101:

CCGCGGCATTGATACCTCTACCCCGT (sense primer for AMY cassette) (SEQ IDNO:27)

amy102:

GCGGCCGCCTGCAATCCTGGATCCACCG (antisense primer for AMY cassette) (SEQ IDNO: 28)

PCR conditions were as follows: 25 cycles of 15 seconds at 94° C., 30seconds at 55° C. and 2 minutes at 72° C. HF polymerase (Clontech) andchromosomal DNA were used as PCR polymerase and PCR template,respectively. The yielded PCR fragment was cloned into pCR2.1-TOPO. As aresult of restriction and sequencing analysis, it was found that theclone which the had correct sequence was obtained. One clone wasselected for further study (pAMY 1113).

3) Construction of an Expression Vector for the AST Gene WhichFunctioned in Phaffia rhodozyma

An expression plasmid for the AST gene was constructed by restrictiondigestion and ligation of each genetic component. At first, a 0.3 kbKpnI/PstI fragment from pTPIT1104 and 1.7 kb SacI/KpnI fragment frompG418Sa5112 were ligated into a pGEM-T plasmid which was digested bySacI and PstI. It was found that 9 clones among 12 transformants hadcorrect structure as a result of restriction digestion and one of thosewas selected for further study (pTPITG1120).

Next, PCR cloning of the AST gene was performed to add the appropriaterestriction site to both ends. PCR primers whose sequences are listed inTABLE 13 were synthesized.

Table 13

PCR primers used to clone the entire AST gene cassette

ast11:

TCTAGAATGTTCATCTTGGTCTTGCTCA (sense primer) (SEQ ID NO: 29)

ast12:

CCTGCAGGTCATTCGACCGGCTTGACCT (antisense primer) (SEQ ID NO: 30)

PCR conditions were as follows: 25 cycles of 15 seconds at 94° C., 30seconds at 55° C. and 2 minutes at 72° C. HF polymerase (Clontech) andpAST1207 were used as PCR polymerase and PCR template, respectively. Theyielded PCR fragment was cloned into pCR2.1-TOPO. As a result ofrestriction and sequencing analysis, it was found that one clone whichhad correct sequence was obtained. This clone was selected for furtherstudy (pAST 113).

Finally, a 1.6 kb SacII/NotI fragment from pAMY1113, a 0.3 kb NotI/XbaIfragment from pTPIP1104 and a 1.5 kb XbaI/Sse83871 fragment from pAST113were ligated into pTPITG1120 which was digested by SacII and Sse83871.It was confirmed that all five transformants tested had correctstructure as a result of restriction analysis. One transformant wasselected for further study (pAATG123).

4) Restoration of Astaxanthin Production in Beta-carotene-producingPhaffia rhodozyma

The expression plasmid for the AST gene (pAATG123) was transformed intobeta-carotene-producing Phaffia rhodozyma ATCC96815. Biolistictransformation was performed as described in Example 3. Twogeneticin-resistant colonies which colored red were picked up andselected for further study. In order to confirm the integration at theAMY locus on the chromosome of P. rhodozyma, a PCR primer whose sequenceis listed in TABLE 14 was synthesized.

Table 14

PCR primer used to confirm the integration of expression plasmid at AMYlocus on the chromosome of P. rhodozyma

amy5:

CTCTCCTGTTCACAAAAACA (sense primer) (SEQ ID NO: 31)

Chromosomal DNA was prepared from those transformants and used as a PCRtemplate. PCR condition were as follows: 25 cycles of 15 seconds at 94°C., 30 seconds at 55° C. and 2 minutes at 72° C. ExTaq polymerase(Takara Shuzo) was used as PCR polymerase. A positive 2.0 kb PCR bandwas yielded in the PCR reaction in which the chromosome obtained fromthe red-colored transformants was used as a template DNA. No PCR bandwas observed in the PCR reaction mixture in which chromosome derivedfrom host strain, P. rhodozyma ATCC96815 was used as a PCR template.

5) Flask Fermentation By Recombinants in Which the Recombinant AST GeneWas Integrated on the Chromosome of Beta-carotene-producing P. rhodozyma

The productivity of astaxanthin was evaluated in the flask fermentation.The medium formulation for flask fermentation is as follows.

TABLE 15 Seed medium formulation for flask fermentation Glucose 30.0 g/lNH₄Cl 4.83 g/l KH₂PO₄ 1.0 g/l MgSO₄-7H₂O 0.88 g/l NaCl 0.06 g/lCaCl₂-2H₂O 0.2 g/l KH phthalate 20.0 g/l FeSO₄-7H₂O 28 mg/l Citricacid-1H₂O 15.0 mg/l ZnSO₄-7H₂O 40.0 mg/l CuSO₄-5H₂O 0.75 mg/lMnSO4-4,5H₂O 0.6 mg/l H₃BO₃ 0.6 mg/l Na₂MoO₄-2H₂O 0.6 mg/l KI 0.15 mg/lMyo-inositol 60.0 mg/l Nicotinic acid 3.0 mg/l Ca D-pantothenate 3.0mg/l Vitamin B1 (thiamin HCl) 3.0 mg/l p-Aminobenzoic acid 1.8 mg/lVitamin B6 (pyridoxine HCl) 0.3 mg/l Biotin 0.048 mg/l 7 ml/Test Tube(21 mm diameter)

TABLE 16 Medium formulation for flask fermentation MgSO₄-7H₂O 2.1 g/lCaCl₂-2H₂O 0.865 g/l (NH₄)₂SO₄ 3.7 g/l FeSO₄-7H₂O 0.28 g/l Glucose(sterilized separately) 22 g/l KH₂PO₄ (sterilized separately) 14.25 g/lCitric acid-1H₂O 0.21 g/l ZnSO₄-7H₂O 70.14 mg/l CuSO₄-5H₂O 10.5 mg/lMnSO4-4,5H₂O 8.4 mg/l H₃BO₃ 8.4 mg/l Na₂MoO₄-2H₂O 8.4 mg/l KI 2.1 mg/lMyo-inositol 0.374 g/l Nicotinic acid 18.7 mg/l Ca D-pantothenate 28.05mg/l Vitamin B1 (thiamin HCl) 18.7 mg/l p-Aminobenzoic acid 11.22 mg/lVitamin B6 (pyridoxine HCl) 1.87 mg/l Biotin 1.122 mg/ CaCO3 10 g/l

1 drop of Actcol (Takeda Chemical Industries Ltd., Osaka, JAPAN) wasadded to each flask.

50 ml (final volume with 5% of seed inoculum) was added per 500 ml flaskwith buffles

Cells were harvested from fermented broth after a 7-day fermentation andanalyzed for their accumulation of astaxanthin and beta-carotene by HPLCas described in Example 4. Results are summarized in TABLE 17.

TABLE 17 Restoration of astaxanthin production by the recombinants inwhich the AST gene was integrated. (Data is indicated as relative titerof astaxanthin and beta-carotene against the titer of beta-caroteneaccumulated by P. rhodozyma ATCC96815) Relative titer (%) StrainAstaxanthin Beta-carotene ATCC96815 :: pR16 34.0% 18.6% ATCC96815 ::pAATG123 16.3% 56.3% ATCC96815   0%  100%

Partial restoration of astaxanthin production by ATCC96815::pAATG123indicated that promoter strength by TPI promoter is not strong enoughfor perfect restoration of astaxanthin production.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodifications are intended to be included within the scope of thefollowing claims.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 32 <210> SEQ ID NO 1 <211>LENGTH: 557 <212> TYPE: PRT <213> ORGANISM: Phaffia rhodozyma <220>FEATURE: <221> NAME/KEY: TRANSIT <222> LOCATION: (1)..(26) <400>SEQUENCE: 1 Met Phe Ile Leu Val Leu Leu Thr Gly Ala Leu Gly Leu Ala AlaPhe 1 5 10 15 Ser Trp Ala Ser Ile Ala Phe Phe Ser Leu Tyr Leu Ala ProArg Arg 20 25 30 Ser Ser Leu Tyr Asn Leu Gln Gly Pro Asn His Thr Asn TyrPhe Thr 35 40 45 Gly Asn Phe Leu Asp Ile Leu Ser Ala Arg Thr Gly Glu GluHis Ala 50 55 60 Lys Tyr Arg Glu Lys Tyr Gly Ser Thr Leu Arg Phe Ala GlyIle Ala 65 70 75 80 Gly Ala Pro Val Leu Asn Ser Thr Asp Pro Lys Val PheAsn His Val 85 90 95 Met Lys Glu Ala Tyr Asp Tyr Pro Lys Pro Gly Met AlaAla Arg Val 100 105 110 Leu Arg Ile Ala Thr Gly Asp Gly Val Val Thr AlaGlu Gly Glu Ala 115 120 125 His Lys Arg His Arg Arg Ile Met Ile Pro SerLeu Ser Ala Gln Ala 130 135 140 Val Lys Ser Met Val Pro Ile Phe Leu GluLys Gly Met Glu Leu Val 145 150 155 160 Asp Lys Met Met Glu Asp Ala AlaGlu Lys Asp Met Ala Val Gly Glu 165 170 175 Ser Ala Gly Glu Lys Lys AlaThr Arg Leu Glu Thr Glu Gly Val Asp 180 185 190 Val Lys Asp Trp Val GlyArg Ala Thr Leu Asp Val Met Ala Leu Ala 195 200 205 Gly Phe Asp Tyr LysSer Asp Ser Leu Gln Asn Lys Thr Asn Glu Leu 210 215 220 Tyr Val Ala PheVal Gly Leu Thr Asp Gly Phe Ala Pro Thr Leu Asp 225 230 235 240 Ser PheLys Ala Ile Met Trp Asp Phe Val Pro Tyr Phe Arg Thr Met 245 250 255 LysArg Arg His Glu Ile Pro Leu Thr Gln Gly Leu Ala Val Ser Arg 260 265 270Arg Val Gly Ile Glu Leu Met Glu Gln Lys Lys Gln Ala Val Leu Gly 275 280285 Ser Ala Ser Asp Gln Ala Val Asp Lys Lys Asp Val Gln Gly Arg Asp 290295 300 Ile Leu Ser Leu Leu Val Arg Ala Asn Ile Ala Ala Asn Leu Pro Glu305 310 315 320 Ser Gln Lys Leu Ser Asp Glu Glu Val Leu Ala Gln Ile SerAsn Leu 325 330 335 Leu Phe Ala Gly Tyr Glu Thr Ser Ser Thr Val Leu ThrTrp Met Phe 340 345 350 His Arg Leu Ser Glu Asp Lys Ala Val Gln Asp LysLeu Arg Glu Glu 355 360 365 Ile Cys Gln Ile Asp Thr Asp Met Pro Thr LeuAsp Glu Leu Asn Ala 370 375 380 Leu Pro Tyr Leu Glu Ala Phe Val Lys GluSer Leu Arg Leu Asp Pro 385 390 395 400 Pro Ser Pro Tyr Ala Asn Arg GluCys Leu Lys Asp Glu Asp Phe Ile 405 410 415 Pro Leu Ala Glu Pro Val IleGly Arg Asp Gly Ser Val Ile Asn Glu 420 425 430 Val Arg Ile Thr Lys GlyThr Met Val Met Leu Pro Leu Phe Asn Ile 435 440 445 Asn Arg Ser Lys PheIle Tyr Gly Glu Asp Ala Glu Glu Phe Arg Pro 450 455 460 Glu Arg Trp LeuGlu Asp Val Thr Asp Ser Leu Asn Ser Ile Glu Ala 465 470 475 480 Pro TyrGly His Gln Ala Ser Phe Ile Ser Gly Pro Arg Ala Cys Phe 485 490 495 GlyTrp Arg Phe Ala Val Ala Glu Met Lys Ala Phe Leu Phe Val Thr 500 505 510Leu Arg Arg Val Gln Phe Glu Pro Ile Ile Ser His Pro Glu Tyr Glu 515 520525 His Ile Thr Leu Ile Ile Ser Arg Pro Arg Ile Val Gly Arg Glu Lys 530535 540 Glu Gly Tyr Gln Met Arg Leu Gln Val Lys Pro Val Glu 545 550 555<210> SEQ ID NO 2 <211> LENGTH: 1932 <212> TYPE: DNA <213> ORGANISM:Phaffia rhodozyma <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(33)..(1706) <221> NAME/KEY: polyA_site <222> LOCATION: (1871) <221>NAME/KEY: mRNA <222> LOCATION: (14)..(1891) <400> SEQUENCE: 2 gaattcggcacgaggccacc tactttctcc at atg ttc atc ttg gtc ttg ctc 53 Met Phe Ile LeuVal Leu Leu 1 5 aca ggt gct tta ggc ctg gct gct ttc tca tgg gca tcc atagcg ttc 101 Thr Gly Ala Leu Gly Leu Ala Ala Phe Ser Trp Ala Ser Ile AlaPhe 10 15 20 ttc agt ctt tac ctc gct ccg agg cga tct tca ctg tat aac cttcag 149 Phe Ser Leu Tyr Leu Ala Pro Arg Arg Ser Ser Leu Tyr Asn Leu Gln25 30 35 ggc ccg aat cat acc aac tac ttt aca ggc aat ttt tta gac atc ctc197 Gly Pro Asn His Thr Asn Tyr Phe Thr Gly Asn Phe Leu Asp Ile Leu 4045 50 55 tca gct cgt aca ggt gaa gag cat gcg aag tac aga gaa aaa tac gga245 Ser Ala Arg Thr Gly Glu Glu His Ala Lys Tyr Arg Glu Lys Tyr Gly 6065 70 agc acc ctc cgg ttt gct ggg atc gct gga gca ccc gtc ttg aac tcg293 Ser Thr Leu Arg Phe Ala Gly Ile Ala Gly Ala Pro Val Leu Asn Ser 7580 85 acc gat ccg aaa gtc ttc aac cat gtg atg aaa gaa gcc tac gac tat341 Thr Asp Pro Lys Val Phe Asn His Val Met Lys Glu Ala Tyr Asp Tyr 9095 100 ccg aaa cct ggt atg gcc gct cga gtg ctc aga att gct acc gga gat389 Pro Lys Pro Gly Met Ala Ala Arg Val Leu Arg Ile Ala Thr Gly Asp 105110 115 ggt gtt gtt acg gcg gaa ggt gaa gct cat aag cga cat cga agg atc437 Gly Val Val Thr Ala Glu Gly Glu Ala His Lys Arg His Arg Arg Ile 120125 130 135 atg atc ccc tct ctg tcc gct cag gcc gtt aag tcg atg gtc ccaatt 485 Met Ile Pro Ser Leu Ser Ala Gln Ala Val Lys Ser Met Val Pro Ile140 145 150 ttc tta gaa aaa ggt atg gaa ctt gtc gac aag atg atg gag gatgcg 533 Phe Leu Glu Lys Gly Met Glu Leu Val Asp Lys Met Met Glu Asp Ala155 160 165 gct gag aag gat atg gcc gtg gga gag tcg gcc ggt gaa aag aaggca 581 Ala Glu Lys Asp Met Ala Val Gly Glu Ser Ala Gly Glu Lys Lys Ala170 175 180 acc aga ctc gag acc gaa gga gtc gat gta aag gat tgg gtc ggtcga 629 Thr Arg Leu Glu Thr Glu Gly Val Asp Val Lys Asp Trp Val Gly Arg185 190 195 gct act ctg gac gtc atg gct ctt gca gga ttt gac tat aag agcgac 677 Ala Thr Leu Asp Val Met Ala Leu Ala Gly Phe Asp Tyr Lys Ser Asp200 205 210 215 tcg ctc cag aac aag acc aat gag ctc tat gtc gct ttt gtcgga ctt 725 Ser Leu Gln Asn Lys Thr Asn Glu Leu Tyr Val Ala Phe Val GlyLeu 220 225 230 acc gat ggg ttt gct cct acc ttg gac tcg ttc aag gct atcatg tgg 773 Thr Asp Gly Phe Ala Pro Thr Leu Asp Ser Phe Lys Ala Ile MetTrp 235 240 245 gat ttt gta cct tac ttc cga act atg aaa cgg aga cat gagata cct 821 Asp Phe Val Pro Tyr Phe Arg Thr Met Lys Arg Arg His Glu IlePro 250 255 260 ttg act caa gga tta gca gtt tcc cga cga gtt ggg atc gagctt atg 869 Leu Thr Gln Gly Leu Ala Val Ser Arg Arg Val Gly Ile Glu LeuMet 265 270 275 gag caa aag aag cag gcc gtg ctt ggc tca gct tcc gat caggct gtt 917 Glu Gln Lys Lys Gln Ala Val Leu Gly Ser Ala Ser Asp Gln AlaVal 280 285 290 295 gat aaa aag gat gtt caa ggt cgg gat atc cta agt ctccta gtg aga 965 Asp Lys Lys Asp Val Gln Gly Arg Asp Ile Leu Ser Leu LeuVal Arg 300 305 310 gca aac atc gcc gcc aac ctg cct gaa tct caa aag ctgtcc gat gag 1013 Ala Asn Ile Ala Ala Asn Leu Pro Glu Ser Gln Lys Leu SerAsp Glu 315 320 325 gag gta ctc gct cag atc agt aac ctg tta ttt gct ggatat gaa act 1061 Glu Val Leu Ala Gln Ile Ser Asn Leu Leu Phe Ala Gly TyrGlu Thr 330 335 340 tct tcg aca gtc ttg aca tgg atg ttt cac cga ctc tcagaa gac aaa 1109 Ser Ser Thr Val Leu Thr Trp Met Phe His Arg Leu Ser GluAsp Lys 345 350 355 gcc gtt cag gat aaa ctt cga gaa gaa att tgt cag atcgac acg gat 1157 Ala Val Gln Asp Lys Leu Arg Glu Glu Ile Cys Gln Ile AspThr Asp 360 365 370 375 atg cct acg cta gac gaa ctt aat gcg ttg cct tatctc gaa gcg ttt 1205 Met Pro Thr Leu Asp Glu Leu Asn Ala Leu Pro Tyr LeuGlu Ala Phe 380 385 390 gtt aag gag tct ctt cgt cta gac cct cct agt ccgtat gct aac cgt 1253 Val Lys Glu Ser Leu Arg Leu Asp Pro Pro Ser Pro TyrAla Asn Arg 395 400 405 gaa tgc tta aag gat gaa gac ttc atc cca ctt gccgag cct gtc att 1301 Glu Cys Leu Lys Asp Glu Asp Phe Ile Pro Leu Ala GluPro Val Ile 410 415 420 ggt cga gat ggg tcg gtc atc aac gag gtc cgg atcacg aaa gga acg 1349 Gly Arg Asp Gly Ser Val Ile Asn Glu Val Arg Ile ThrLys Gly Thr 425 430 435 atg gtc atg ctt ccg ttg ttc aac atc aat cgt tcaaag ttc att tat 1397 Met Val Met Leu Pro Leu Phe Asn Ile Asn Arg Ser LysPhe Ile Tyr 440 445 450 455 gga gaa gat gca gaa gaa ttc aga ccg gag aggtgg ctt gag gac gta 1445 Gly Glu Asp Ala Glu Glu Phe Arg Pro Glu Arg TrpLeu Glu Asp Val 460 465 470 aca gac tcg ctc aac agt att gaa gca ccc tatgga cac cag gcg agc 1493 Thr Asp Ser Leu Asn Ser Ile Glu Ala Pro Tyr GlyHis Gln Ala Ser 475 480 485 ttt atc tct gga ccc aga gct tgc ttt ggt tggcga ttt gct gtc gcc 1541 Phe Ile Ser Gly Pro Arg Ala Cys Phe Gly Trp ArgPhe Ala Val Ala 490 495 500 gag atg aag gcc ttc ttg ttt gtc act ctc cgtcgg gtc cag ttc gag 1589 Glu Met Lys Ala Phe Leu Phe Val Thr Leu Arg ArgVal Gln Phe Glu 505 510 515 ccc atc atc tct cat cca gag tac gag cac atcacc ttg atc att tcc 1637 Pro Ile Ile Ser His Pro Glu Tyr Glu His Ile ThrLeu Ile Ile Ser 520 525 530 535 cgt cct cga atc gtt ggt aga gag aag gagggg tac cag atg cgt ttg 1685 Arg Pro Arg Ile Val Gly Arg Glu Lys Glu GlyTyr Gln Met Arg Leu 540 545 550 cag gtc aag ccg gtc gaa tga gttgattcttcatatgttaa gagaagttct 1736 Gln Val Lys Pro Val Glu 555 atatctgagaatgtgtgact aggacaatgc cttctttgta tcgatttgtt tctcataccc 1796 gggcaggcgctatgacttct acgtcgtcta tcgtcgctct ggactctctt cttaccctat 1856 atattattccatccgaaaaa aaaaaaaaaa aaaaaaaaaa aaaaagcggc cgctcgagcc 1916 ggctcgtgccgaattc 1932 <210> SEQ ID NO 3 <211> LENGTH: 557 <212> TYPE: PRT <213>ORGANISM: Phaffia rhodozyma <400> SEQUENCE: 3 Met Phe Ile Leu Val LeuLeu Thr Gly Ala Leu Gly Leu Ala Ala Phe 1 5 10 15 Ser Trp Ala Ser IleAla Phe Phe Ser Leu Tyr Leu Ala Pro Arg Arg 20 25 30 Ser Ser Leu Tyr AsnLeu Gln Gly Pro Asn His Thr Asn Tyr Phe Thr 35 40 45 Gly Asn Phe Leu AspIle Leu Ser Ala Arg Thr Gly Glu Glu His Ala 50 55 60 Lys Tyr Arg Glu LysTyr Gly Ser Thr Leu Arg Phe Ala Gly Ile Ala 65 70 75 80 Gly Ala Pro ValLeu Asn Ser Thr Asp Pro Lys Val Phe Asn His Val 85 90 95 Met Lys Glu AlaTyr Asp Tyr Pro Lys Pro Gly Met Ala Ala Arg Val 100 105 110 Leu Arg IleAla Thr Gly Asp Gly Val Val Thr Ala Glu Gly Glu Ala 115 120 125 His LysArg His Arg Arg Ile Met Ile Pro Ser Leu Ser Ala Gln Ala 130 135 140 ValLys Ser Met Val Pro Ile Phe Leu Glu Lys Gly Met Glu Leu Val 145 150 155160 Asp Lys Met Met Glu Asp Ala Ala Glu Lys Asp Met Ala Val Gly Glu 165170 175 Ser Ala Gly Glu Lys Lys Ala Thr Arg Leu Glu Thr Glu Gly Val Asp180 185 190 Val Lys Asp Trp Val Gly Arg Ala Thr Leu Asp Val Met Ala LeuAla 195 200 205 Gly Phe Asp Tyr Lys Ser Asp Ser Leu Gln Asn Lys Thr AsnGlu Leu 210 215 220 Tyr Val Ala Phe Val Gly Leu Thr Asp Gly Phe Ala ProThr Leu Asp 225 230 235 240 Ser Phe Lys Ala Ile Met Trp Asp Phe Val ProTyr Phe Arg Thr Met 245 250 255 Lys Arg Arg His Glu Ile Pro Leu Thr GlnGly Leu Ala Val Ser Arg 260 265 270 Arg Val Gly Ile Glu Leu Met Glu GlnLys Lys Gln Ala Val Leu Gly 275 280 285 Ser Ala Ser Asp Gln Ala Val AspLys Lys Asp Val Gln Gly Arg Asp 290 295 300 Ile Leu Ser Leu Leu Val ArgAla Asn Ile Ala Ala Asn Leu Pro Glu 305 310 315 320 Ser Gln Lys Leu SerAsp Glu Glu Val Leu Ala Gln Ile Ser Asn Leu 325 330 335 Leu Phe Ala GlyTyr Glu Thr Ser Ser Thr Val Leu Thr Trp Met Phe 340 345 350 His Arg LeuSer Glu Asp Lys Ala Val Gln Asp Lys Leu Arg Glu Glu 355 360 365 Ile CysGln Ile Asp Thr Asp Met Pro Thr Leu Asp Glu Leu Asn Ala 370 375 380 LeuPro Tyr Leu Glu Ala Phe Val Lys Glu Ser Leu Arg Leu Asp Pro 385 390 395400 Pro Ser Pro Tyr Ala Asn Arg Glu Cys Leu Lys Asp Glu Asp Phe Ile 405410 415 Pro Leu Ala Glu Pro Val Ile Gly Arg Asp Gly Ser Val Ile Asn Glu420 425 430 Val Arg Ile Thr Lys Gly Thr Met Val Met Leu Pro Leu Phe AsnIle 435 440 445 Asn Arg Ser Lys Phe Ile Tyr Gly Glu Asp Ala Glu Glu PheArg Pro 450 455 460 Glu Arg Trp Leu Glu Asp Val Thr Asp Ser Leu Asn SerIle Glu Ala 465 470 475 480 Pro Tyr Gly His Gln Ala Ser Phe Ile Ser GlyPro Arg Ala Cys Phe 485 490 495 Gly Trp Arg Phe Ala Val Ala Glu Met LysAla Phe Leu Phe Val Thr 500 505 510 Leu Arg Arg Val Gln Phe Glu Pro IleIle Ser His Pro Glu Tyr Glu 515 520 525 His Ile Thr Leu Ile Ile Ser ArgPro Arg Ile Val Gly Arg Glu Lys 530 535 540 Glu Gly Tyr Gln Met Arg LeuGln Val Lys Pro Val Glu 545 550 555 <210> SEQ ID NO 4 <211> LENGTH: 3969<212> TYPE: DNA <213> ORGANISM: Phaffia rhodozyma <220> FEATURE: <221>NAME/KEY: 5′UTR <222> LOCATION: (517)..(518) <221> NAME/KEY: intron<222> LOCATION: (784)..(898) <221> NAME/KEY: intron <222> LOCATION:(1016)..(1087) <221> NAME/KEY: intron <222> LOCATION: (1180)..(1302)<221> NAME/KEY: intron <222> LOCATION: (1518)..(1600) <221> NAME/KEY:intron <222> LOCATION: (1635)..(1723) <221> NAME/KEY: intron <222>LOCATION: (1867)..(1939) <221> NAME/KEY: intron <222> LOCATION:(2000)..(2081) <221> NAME/KEY: intron <222> LOCATION: (2182)..(2257)<221> NAME/KEY: intron <222> LOCATION: (2355)..(2431) <221> NAME/KEY:intron <222> LOCATION: (2543)..(2618) <221> NAME/KEY: intron <222>LOCATION: (2653)..(2742) <221> NAME/KEY: intron <222> LOCATION:(2815)..(2962) <221> NAME/KEY: intron <222> LOCATION: (3051)..(3113)<221> NAME/KEY: intron <222> LOCATION: (3172)..(3247) <221> NAME/KEY:intron <222> LOCATION: (3322)..(3398) <221> NAME/KEY: intron <222>LOCATION: (3424)..(3513) <221> NAME/KEY: polyA_site <222> LOCATION:(3865)..(3866) <221> NAME/KEY: intron <222> LOCATION: (653)..(734) <400>SEQUENCE: 4 cggaccgaag cctcgccagc agttgatcaa gcgaaccaag ccgaacaatcctggcgcgcc 60 tggaggagcg ggagcgggag gagcagcagg tgatgcatcg ggtggacagaatcagtagtg 120 tgtgtgtatg tgtgtagtgt agttgggttg tcccatgtgc ttcttcttatcatcatcatt 180 tctttaaaat ctctacattg aatgtttacc ggaacgggct ttgatgatactacggaccac 240 gttgtgtaac cagttcgatt gagattacga ttagatagcc gatccgtcgatcagatctcg 300 atctagagcg acatctggct cgatcggtcc ttgccgaaaa tcagggcaccgatcagggca 360 gaggaacgcc gaggccgaac gagacagaca caccatcatc atcagccatgtcttttttgt 420 gatcgttttt acatactacc cgtcgattct aaccttcttt cttcttctcttgccatcttt 480 gcattctcta tctcgtgtaa catcgatccg attcttgcca cctactttctccatatgttc 540 atcttggtct tgctcacagg tgctttaggc ctggctgctt tctcatgggcatccatagcg 600 ttcttcagtc tttacctcgc tccgaggcga tcttcactgt ataaccttcagggtaagaat 660 tgagctctgg aatcatgctt gtgtaaatcc tataatctca ttcatcctattcctcttctt 720 catcctctct tcaggcccga atcataccaa ctactttaca ggcaattttttagacatcct 780 ctcgtgagtt ttcatcattg gctcagtcgt ccaatcttaa cgatcatcgctaacgacctt 840 tcggacgcgt tcttctttct atgtgaaatc tgatctttgg tttgttacgagagcacagag 900 ctcgtacagg tgaagagcat gcgaagtaca gagaaaaata cggaagcaccctccggtttg 960 ctgggatcgc tggagcaccc gtcttgaact cgaccgatcc gaaagtcttcaaccagtttg 1020 tccatccgaa ccctcatcct cctctgctga tcaattcaac tgtagttaacgcactttgaa 1080 tggacagtgt gatgaaagaa gcctacgact atccgaaacc tggtatggccgctcgagtgc 1140 tcagaattgc taccggagat ggtgttgtta cggcggaagg tgcttttcaagttctcttat 1200 atcacatcta atccactcgg cgcgattgaa ctcaacattt ctgacgagcctgtcaccttg 1260 ttttcacttc atggtctcgg tgcatcttgt ctcatctcat aggtgaagctcataagcgac 1320 atcgaaggat catgatcccc tctctgtccg ctcaggccgt taagtcgatggtcccaattt 1380 tcttagaaaa aggtatggaa cttgtcgaca agatgatgga ggatgcggctgagaaggata 1440 tggccgtggg agagtcggcc ggtgaaaaga aggcaaccag actcgagaccgaaggagtcg 1500 atgtaaagga ttgggtcgtg agtacccgcc tattccttca ccttgatggacgaagcatat 1560 caaggaaagg ttcattgact gacaaacact atcttaccag ggtcgagctactctggacgt 1620 catggctctt gcaggtcagt ctactctctc ttataaatgc tccacatatgtatgcatgta 1680 ctgacatgct cttcctatat tcgatacgac gtcatatgtc caggatttgactataagagc 1740 gactcgctcc agaacaagac caatgagctc tatgtcgctt ttgtcggacttaccgatggg 1800 tttgctccta ccttggactc gttcaaggct atcatgtggg attttgtaccttacttccga 1860 actatggtat gtctgccatt ctttgatatc caaagattat ggataggttacttgctaaaa 1920 tttcacctat cgtgaacaga aacggagaca tgagatacct ttgactcaaggattagcagt 1980 ttcccgacga gttgggatcg taagtgccag atcaagcctc tctgaatattcttggtcatc 2040 atcttaacct cctaggctca ttcatccatg gtgcgcaata ggagcttatggagcaaaaga 2100 agcaggccgt gcttggctca gcttccgatc aggctgttga taaaaaggatgttcaaggtc 2160 gggatatcct aagtctccta ggttagtaac gtttttaaac gtatatacagagcggcgaca 2220 ttctttccct gacaactgtc aacatgctcg ttactagtga gagcaaacatcgccgccaac 2280 ctgcctgaat ctcaaaagct gtccgatgag gaggtactcg ctcagatcagtaacctgtta 2340 tttgctggat atgagtgtgt atcctttccc ctctctatcc ttagctgattaaaagcacta 2400 atagaggtct ttatgtttcc tgtttgatca gaacttcttc gacagtcttgacatggatgt 2460 ttcaccgact ctcagaagac aaagccgttc aggataaact tcgagaagaaatttgtcaga 2520 tcgacacgga tatgcctacg ctgtgaggat gtttttgatg ctaaattacttcttcttgca 2580 aatgactaaa acggccttcc attcttgatc cattttagag acgaacttaatgcgttgcct 2640 tatctcgaag cggttggttc tcgattcttg gtcttgtctt ccaaatacaatacggattat 2700 tgctcatctg atttgcgtct acgggctgtg gaatttaact agtttgttaaggagtctctt 2760 cgtctagacc ctcctagtcc gtatgctaac cgtgaatgct taaaggatgaagacgtatgt 2820 tggcttcatc acgcataatt ttcatttcat attcctttgt acatacgcatacaggctgac 2880 cgagctcaaa ttccggcttc ctcttctgtg cttctttttc tggcctttcttatcttcatt 2940 cttcaaccaa aatttgtcac agttcatccc acttgccgag cctgtcattggtcgagatgg 3000 gtcggtcatc aacgaggtcc ggatcacgaa aggaacgatg gtcatgcttcgtaagttttc 3060 ctttatttca tctcgtccat gaaatagttt ctgatagacg cggaccaattcagcgttgtt 3120 caacatcaat cgttcaaagt tcatttatgg agaagatgca gaagaattcaggtacaattc 3180 gttttctttt aaaagccaat cggtttcgta tcgtaattga ccgggctctcttttaatttc 3240 tcgaaagacc ggagaggtgg cttgaggacg taacagactc gctcaacagtattgaagcac 3300 cctatggaca ccaggcgagc tgtatgtttt attgatttta tctttgtgaattttgcaaaa 3360 cgttgaactt cgcgcttccc ttgttgttga aatcccagtt atctctggacccagagcttg 3420 cttgtaagtt tcttctcatc tggcgcctta gcagtatccg atcagccatctagttctttg 3480 tacgattgtt tctgactctc tcgactttcg cagtggttgg cgatttgctgtcgccgagat 3540 gaaggccttc ttgtttgtca ctctccgtcg ggtccagttc gagcccatcatctctcatcc 3600 agagtacgag cacatcacct tgatcatttc ccgtcctcga atcgttggtagagagaagga 3660 ggggtaccag atgcgtttgc aggtcaagcc ggtcgaatga gttgattcttcatatgttaa 3720 gagaagttct atatctgaga atgtgtgact aggacaatgc cttctttgtatcgatttgtt 3780 tctcataccc gggcaggcgc tatgacttct acgtcgtcta tcgtcgctctggactctctt 3840 cttaccctat atattattcc atccgtctgt atatttgtct atcacgacgtctgtgtcgtc 3900 aactcaatat tcagcctctt catgcttctg tgtctccata gatgtgatcttcatgtttgt 3960 cgactgcag 3969 <210> SEQ ID NO 5 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Sense primer forexpression of the AST gene in E. coli <400> SEQUENCE: 5 gttcaaagttcatttatgga 20 <210> SEQ ID NO 6 <211> LENGTH: 47 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Antisense primer for expession ofthe AST gene in E. coli <400> SEQUENCE: 6 ggatcctcag tggtggtggtggtggtgttc gaccggcttg acctgca 47 <210> SEQ ID NO 7 <211> LENGTH: 45<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: 5′ sense primerfor expression of a modified AST gene in E. coli <400> SEQUENCE: 7catatgcacc accaccacca ccacctgtat aaccttcagg ggccc 45 <210> SEQ ID NO 8<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: 5′ antisense primer for expression of a modified AST gene inE. coli <400> SEQUENCE: 8 gtaacaacac catctccggt 20 <210> SEQ ID NO 9<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: 3′ anti sense primer for expression of a modified AST gene inE. coli <400> SEQUENCE: 9 ggatcctcaa ctcattcgac cggctt 26 <210> SEQ IDNO 10 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Genome walking primer for cloning of the AST gene<400> SEQUENCE: 10 tagagagaag gaggggtacc agatgc 26 <210> SEQ ID NO 11<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Antisense primer for cloning of the terminator region of theAST gene <400> SEQUENCE: 11 ccccggattg tggagaaact 20 <210> SEQ ID NO 12<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Sense primer for cloning the genomic AST gene <400> SEQUENCE:12 atgttcatct tggtcttgct 20 <210> SEQ ID NO 13 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Antisense primer forcloning the genomic AST gene <400> SEQUENCE: 13 acgtagaagt catagcgcct 20<210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Sense primer for RT-PCR of the AST gene <400>SEQUENCE: 14 tttgactcaa ggattagcag 20 <210> SEQ ID NO 15 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: Antisenseprimer for RT-PCR of the AST gene <400> SEQUENCE: 15 tgtcttctgagagtcggtga 20 <210> SEQ ID NO 16 <211> LENGTH: 24 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Degenerate sense primer for cloningof the TPI gene <221> NAME/KEY: misc_feature <222> LOCATION: 3 <223>OTHER INFORMATION: n is a or c or g or t <221> NAME/KEY: misc_feature<222> LOCATION: 6 <223> OTHER INFORMATION: n is a or c or g or t <221>NAME/KEY: misc_feature <222> LOCATION: 15 <223> OTHER INFORMATION: n isa or c or g or t <221> NAME/KEY: misc_feature <222> LOCATION: 18 <223>OTHER INFORMATION: n is a or c or g or t <221> NAME/KEY: misc_feature<222> LOCATION: 21 <223> OTHER INFORMATION: n is a or c or g or t <400>SEQUENCE: 16 mgnacnttyt tygtnggngg naay 24 <210> SEQ ID NO 17 <211>LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Degenerate antisense primer for cloning the TPI gene <221> NAME/KEY:misc_feature <222> LOCATION: 3 <223> OTHER INFORMATION: n is a or c or gor t <221> NAME/KEY: misc_feature <222> LOCATION: 6 <223> OTHERINFORMATION: n is a or c or g or t <221> NAME/KEY: misc_feature <222>LOCATION: 9 <223> OTHER INFORMATION: n is a or c or g or t <221>NAME/KEY: misc_feature <222> LOCATION: 12 <223> OTHER INFORMATION: n isa or c or g or t <221> NAME/KEY: misc_feature <222> LOCATION: 18 <223>OTHER INFORMATION: n is a or c or g or t <221> NAME/KEY: misc_feature<222> LOCATION: 24 <223> OTHER INFORMATION: n is a or c or g or t <400>SEQUENCE: 17 gcnccnccna cnarraancc rtcnacrtc 29 <210> SEQ ID NO 18 <211>LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Primary walking primer for cloning of the TPI terminator <400> SEQUENCE:18 gcttacctcg cttccaacgt ttcccag 27 <210> SEQ ID NO 19 <211> LENGTH: 27<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Nested walkingprimer for cloning of the TPI terminator <400> SEQUENCE: 19 ggatctgtctctgcctccaa ctgcaag 27 <210> SEQ ID NO 20 <211> LENGTH: 27 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Primary walking primerfor cloning of the TPI promoter <400> SEQUENCE: 20 gggtcaatgt cggcagcgagaagccca 27 <210> SEQ ID NO 21 <211> LENGTH: 27 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Nested walking primer for cloning ofthe TPI promoter <400> SEQUENCE: 21 atgtactcgg tagcactgat caagtag 27<210> SEQ ID NO 22 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Sense primer for construction of the TPIpromoter cassette <400> SEQUENCE: 22 gcggccgcat ccgtctcgcc atcagtct 28<210> SEQ ID NO 23 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Antisense primer for construction of the TPIpromoter cassette <400> SEQUENCE: 23 cctgcaggtc tagagatgaa taaatataaagagt 34 <210> SEQ ID NO 24 <211> LENGTH: 34 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Sense primer for construction of theTPI terminator cassette <400> SEQUENCE: 24 cctgcaggta aatatatccagggattaacc ccta 34 <210> SEQ ID NO 25 <211> LENGTH: 28 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Antisense primer forconstruction of the TPI terminator cassette <400> SEQUENCE: 25ggtacccgtg cgcagtcgac cgagacat 28 <210> SEQ ID NO 26 <211> LENGTH: 35<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Degenerate senseprimer for cloning of the AMY gene <221> NAME/KEY: misc_feature <222>LOCATION: 15 <223> OTHER INFORMATION: n is a or c or g or t <221>NAME/KEY: misc_feature <222> LOCATION: 21 <223> OTHER INFORMATION: n isa or c or g or t <221> NAME/KEY: misc_feature <222> LOCATION: 27 <223>OTHER INFORMATION: n is a or c or g or t <221> NAME/KEY: misc_feature<222> LOCATION: 30 <223> OTHER INFORMATION: n is a or c or g or t <400>SEQUENCE: 26 gaytayathc arggnatggg nttyrmngcn athtg 35 <210> SEQ ID NO27 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Degenerate antisense primer for cloning of the AMY gene <221>NAME/KEY: misc_feature <222> LOCATION: 6 <223> OTHER INFORMATION: n is aor c or g or t <221> NAME/KEY: misc_feature <222> LOCATION: 9 <223>OTHER INFORMATION: n is a or c or g or t <221> NAME/KEY: misc_feature<222> LOCATION: 24 <223> OTHER INFORMATION: n is a or c or g or t <221>NAME/KEY: misc_feature <222> LOCATION: 30 <223> OTHER INFORMATION: n isa or c or g or t <400> SEQUENCE: 27 tgytcngtnc crtartadat datnggdatnccrtc 35 <210> SEQ ID NO 28 <211> LENGTH: 26 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Sense primer for construction of apartial AMY cassette <400> SEQUENCE: 28 ccgcggcatt gatacctcta ccccgt 26<210> SEQ ID NO 29 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Antisense primer for construction of a partialAMY cassette <400> SEQUENCE: 29 gcggccgcct gcaatcctgg atccaccg 28 <210>SEQ ID NO 30 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Sense primer for construction of the AST cassette<400> SEQUENCE: 30 tctagaatgt tcatcttggt cttgctca 28 <210> SEQ ID NO 31<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Antisense primer for construction of the AST cassette <400>SEQUENCE: 31 cctgcaggtc attcgaccgg cttgacct 28 <210> SEQ ID NO 32 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Sense primer for confirmation of integration at the AMY locus by PCRanalysis <400> SEQUENCE: 32 ctctcctgtt cacaaaaaca 20

What is claimed is:
 1. An isolated polynucleotide encoding 4n enzymederived from P. rhodozyma and having astaxanthin synthetase activitywhich enzyme catalyzes the reaction of beta-carotene to astaxanthin. 2.An isolated polynucleotide according to claim 1 which is selected from(a) a nucleotide sequence which encodes an enzyme having the amino acidsequence shown in SEQ ID NO: 1, or (b) a nucleotide sequence whichhybridizes to the complement of a nucleotide sequence which encodes SEQID NO:1 under the following hybridization conditions: 50% v/v formamide,5×SSC, 2% w/v blocking agent, 0.1% N-lauroylsarcosine, 0.3% SDS it 42°C. overnight and which hybrid has astaxanthin synthetase activity.
 3. Anisolated polynucleotide according to claim 1 which is selected from thegroup consisting of: (i) SEQ ID NO: 2; (ii) a nucleotide sequence which,because of the degeneracy of the genetic code, encodes an astaxanthinsynthetase having the same amino acid sequence as that encoded by SEQ IDNO:2; and (iii) a nucleotide sequence which hybridizes to the complementof the nucleotide sequence from i) or ii) under standard hybridizingconditions (50% v/v formamide, 5×SSC, 2% w/v blocking agent, 0.1%N-lauroylsarcosine, 0.3% SDS at 42° C. overnight).
 4. A isolatedpolynucleotide according to claim 1 which is selected from the groupconsisting of: (i) SEQ ID NO: 3; (ii) a nucleotide sequence which,because of the degeneracy of the genetic code, encodes an astaxanthinsynthetase having the same amino acid sequence as that encoded by SEQ IDNO:3; and (iii) a nucleotide sequence which hybridizes to the complementof the nucleotide sequence from i) or ii) under standard hybridizingconditions (50% v/v formamide, 5×SSC 2% w/v blocking agent, 0.1%N-lauroylsarcosine, 0.3% SDS at 42° C. overnight).
 5. A vector orplasmid comprising a polynucleotide which encodes an enzyme derived fromP. rohodozyma having astaxanthin synthase activity which catalyzes thereaction of beta-carotene to astaxanthin.
 6. A vector or plasmidaccording to claim 5 wherein the polynucleotide encodes SEQ ID NO:1. 7.A vector or plasmid according to claim 5 wherein the polynucleotide isSEQ ID NO:2.
 8. A vector or plasmid according to claim 5 wherein thepolynucleotide is SEQ ID NO:3.
 9. A host cell transformed or transfectedwith a polynucleotide which encodes an enzyme derived from P. rhodozymahaving astaxanthin synthase activity.
 10. A host cell according to claim9 wherein the polynucleotide encodes a polypeptide having the sequenceof SEQ ID NO:1.
 11. A host cell according to claim 9 wherein thepolynucleotide is SEQ ID NO:2.
 12. A host cell according to claim 9wherein the polynucleotide is SEQ ID NO:3.
 13. A host cell according toclaim 9 which is transfected or transformed with a vector or a plasmidcomprising: (a) a polynucleotide which encodes the polypeptide of SEQ IDNO:1; (b) SEQ ID NO:2; or (c) SEQ ID NO:3.
 14. A process for producing apolypeptide derived from P. rhodozyma having astaxanthin synthaseactivity comprising culturing a host cell transformed with apolynucleotide which encodes an enzyme having astaxanthin synthaseactivity under conditions conductive to produce the enzyme.
 15. Aprocess according to claim 14 wherein the polynucleotide encodes thepolypeptide of SEQ ID NO:1.
 16. A process according to claim 14 whereinthe polynucleotide is SEQ ID NO:2.
 17. A process according to claim 14wherein the polynucleotide is SEQ ID NO:3.
 18. An isolatedpolynucleotide encoding a polypeptide which is SEQ ID NO:1.
 19. Anisolated polynucleotide consisting of SEQ ID NO:2.
 20. An isolatedpolynucleotide consisting of SEQ ID NO:3.
 21. A recombinantly-producedpolypeptide having astaxanthin synthase activity which is SEQ ID NO:1.22. A host cell transformed with the vector of claim 21.